Surface plasmon resonance (SPR) is a label-free detection method that has become a reliable platform for clinical analysis of biomolecular interactions. This review discusses the principles, applications, and advancements of SPR and SPR imaging (SPRI) in biosensor technologies. SPR measures refractive index changes in response to biomolecular interactions, enabling real-time, high-sensitivity detection without the need for labels. SPRI enhances this by allowing simultaneous processing of hundreds or thousands of samples, making it suitable for high-throughput screening and multiplex analysis. SPR biosensors are used in biomedical, proteomics, genomics, and bioengineering fields for detecting interactions, conformational changes, and mutations. They are also applied in drug discovery, disease diagnosis, and biomarker detection. SPR signal amplification strategies, such as using metal nanoparticles, magnetic nanoparticles, and carbon-based nanomaterials, improve sensitivity and detection limits. SPR biosensors offer advantages like portability, sensitivity, and the ability to detect multiple targets simultaneously. However, challenges remain in achieving high specificity and sensitivity, particularly for low-concentration biomarkers. SPR technology has expanded its applications to biomedical, environmental, and industrial areas, showing promise in disease diagnosis, drug discovery, and foodborne pathogen detection. The review highlights the potential of SPR and SPRI in advancing biosensing technologies for various biological and medical applications.Surface plasmon resonance (SPR) is a label-free detection method that has become a reliable platform for clinical analysis of biomolecular interactions. This review discusses the principles, applications, and advancements of SPR and SPR imaging (SPRI) in biosensor technologies. SPR measures refractive index changes in response to biomolecular interactions, enabling real-time, high-sensitivity detection without the need for labels. SPRI enhances this by allowing simultaneous processing of hundreds or thousands of samples, making it suitable for high-throughput screening and multiplex analysis. SPR biosensors are used in biomedical, proteomics, genomics, and bioengineering fields for detecting interactions, conformational changes, and mutations. They are also applied in drug discovery, disease diagnosis, and biomarker detection. SPR signal amplification strategies, such as using metal nanoparticles, magnetic nanoparticles, and carbon-based nanomaterials, improve sensitivity and detection limits. SPR biosensors offer advantages like portability, sensitivity, and the ability to detect multiple targets simultaneously. However, challenges remain in achieving high specificity and sensitivity, particularly for low-concentration biomarkers. SPR technology has expanded its applications to biomedical, environmental, and industrial areas, showing promise in disease diagnosis, drug discovery, and foodborne pathogen detection. The review highlights the potential of SPR and SPRI in advancing biosensing technologies for various biological and medical applications.