Electrospinning is a cost-effective and flexible technique for producing nanofibers with large specific surface areas, functionalized surfaces, and stable structures. This review outlines the principles of electrospinning, strategies for producing nanofibers with different diameters, morphologies, and structures, and their applications in electrochemical biosensors. The review also discusses the challenges and future prospects of electrospinning technology, including the need for improved theoretical models, precise control of environmental factors, safety concerns related to solvent volatilization and nanofiber inhalation, miniaturization of biosensors, and the potential of electrospun nanofibers in biomimetics and bionic devices. Electrospun nanofibers have been widely used in electrochemical biosensors for detecting various molecules such as glucose, hydrogen peroxide (H2O2), uric acid (UA), dopamine (DA), ascorbic acid (AA), protein, and amino acids. The review highlights the advantages of electrospun nanofibers in enhancing the sensitivity, selectivity, and stability of biosensors.Electrospinning is a cost-effective and flexible technique for producing nanofibers with large specific surface areas, functionalized surfaces, and stable structures. This review outlines the principles of electrospinning, strategies for producing nanofibers with different diameters, morphologies, and structures, and their applications in electrochemical biosensors. The review also discusses the challenges and future prospects of electrospinning technology, including the need for improved theoretical models, precise control of environmental factors, safety concerns related to solvent volatilization and nanofiber inhalation, miniaturization of biosensors, and the potential of electrospun nanofibers in biomimetics and bionic devices. Electrospun nanofibers have been widely used in electrochemical biosensors for detecting various molecules such as glucose, hydrogen peroxide (H2O2), uric acid (UA), dopamine (DA), ascorbic acid (AA), protein, and amino acids. The review highlights the advantages of electrospun nanofibers in enhancing the sensitivity, selectivity, and stability of biosensors.