Electrospinning is a cost-effective and flexible technique for producing nanofibers with large specific surface areas, functionalized surfaces, and stable structures. It has gained significant attention in electrochemical biosensors due to its excellent morphological and structural properties. This review discusses the principles of electrospinning, strategies for controlling nanofiber morphology and structure, and the application of electrospun nanofibers in electrochemical biosensors. It also addresses the challenges and future prospects of electrospinning technology, including the challenges of large-scale production. Electrospinning involves spinning polymer solutions or melts under a strong electric field, resulting in the formation of nanofibers. The process is simple, cost-effective, and versatile, making it suitable for industrial production. Electrospun nanofibers can be used as substrate materials or functional components in biosensors. They provide a large surface area for enhancing the adsorption of biomolecules, improving sensor sensitivity and detection limits. The pore structure of nanofibers also facilitates the diffusion and transfer of biomolecules, enhancing sensor response speed and stability. The diameter of nanofibers is influenced by factors such as polymer concentration, electric field strength, and the distance between the capillary port and the collector. The morphology and structure of nanofibers can be controlled by adjusting parameters such as solution properties, electric field strength, and environmental conditions. Various strategies have been developed to produce nanofibers with different morphologies, including bead-on-string, porous, hollow, and composite nanofibers. Electrospun nanofibers have been widely applied in electrochemical biosensors for the detection of various molecules, including glucose, hydrogen peroxide, uric acid, dopamine, ascorbic acid, proteins, and amino acids. For example, glucose sensors based on electrospun nanofibers have shown high sensitivity and selectivity. Non-enzyme glucose sensors using transition metals or metal oxides have also been developed, offering advantages such as lower cost and environmental stability. H2O2 sensors have been developed using noble metal nanoparticles and metal oxide nanofibers, demonstrating excellent electrocatalytic activity. Other biomolecules, such as UA, DA, and AA, have also been detected using electrospun nanofibers, with CNFs showing promising performance due to their good dispersibility, wettability, conductivity, and biocompatibility. Despite its advantages, electrospinning technology faces challenges in practical application, including the need for improved theoretical models, precise control of environmental factors, safety issues related to solvent volatilization, and the safety of nanofibers. Additionally, the miniaturization of biosensors is an important challenge for future development. Overall, electrospinning technology holds great potential for the development of advanced electrochemical biosensors and other applications.Electrospinning is a cost-effective and flexible technique for producing nanofibers with large specific surface areas, functionalized surfaces, and stable structures. It has gained significant attention in electrochemical biosensors due to its excellent morphological and structural properties. This review discusses the principles of electrospinning, strategies for controlling nanofiber morphology and structure, and the application of electrospun nanofibers in electrochemical biosensors. It also addresses the challenges and future prospects of electrospinning technology, including the challenges of large-scale production. Electrospinning involves spinning polymer solutions or melts under a strong electric field, resulting in the formation of nanofibers. The process is simple, cost-effective, and versatile, making it suitable for industrial production. Electrospun nanofibers can be used as substrate materials or functional components in biosensors. They provide a large surface area for enhancing the adsorption of biomolecules, improving sensor sensitivity and detection limits. The pore structure of nanofibers also facilitates the diffusion and transfer of biomolecules, enhancing sensor response speed and stability. The diameter of nanofibers is influenced by factors such as polymer concentration, electric field strength, and the distance between the capillary port and the collector. The morphology and structure of nanofibers can be controlled by adjusting parameters such as solution properties, electric field strength, and environmental conditions. Various strategies have been developed to produce nanofibers with different morphologies, including bead-on-string, porous, hollow, and composite nanofibers. Electrospun nanofibers have been widely applied in electrochemical biosensors for the detection of various molecules, including glucose, hydrogen peroxide, uric acid, dopamine, ascorbic acid, proteins, and amino acids. For example, glucose sensors based on electrospun nanofibers have shown high sensitivity and selectivity. Non-enzyme glucose sensors using transition metals or metal oxides have also been developed, offering advantages such as lower cost and environmental stability. H2O2 sensors have been developed using noble metal nanoparticles and metal oxide nanofibers, demonstrating excellent electrocatalytic activity. Other biomolecules, such as UA, DA, and AA, have also been detected using electrospun nanofibers, with CNFs showing promising performance due to their good dispersibility, wettability, conductivity, and biocompatibility. Despite its advantages, electrospinning technology faces challenges in practical application, including the need for improved theoretical models, precise control of environmental factors, safety issues related to solvent volatilization, and the safety of nanofibers. Additionally, the miniaturization of biosensors is an important challenge for future development. Overall, electrospinning technology holds great potential for the development of advanced electrochemical biosensors and other applications.