Surface plasmon resonance (SPR) is a label-free detection method that has emerged as a reliable platform for clinical analysis of biomolecular interactions. This review discusses the principles, applications, and advancements in SPR and surface plasmon resonance imaging (SPRI) in optical-based biosensors. SPR measures refractive index changes near thin metal layers in response to biomolecular interactions, enabling real-time, high-sensitivity measurements without labels. The technique is widely used in biomedical, proteomics, genomics, and bioengineering fields. Key applications include interaction analyses, conformational change studies, mutation detection, high-throughput screening, and cellular analysis. Signal amplification strategies, such as using metal nanoparticles, magnetic nanoparticles, and carbon-based nanomaterials, enhance SPR sensitivity. Recent advances in SPR technology, including improvements in spatial resolution and multiplexing capabilities, continue to expand its utility in various biological and clinical applications.Surface plasmon resonance (SPR) is a label-free detection method that has emerged as a reliable platform for clinical analysis of biomolecular interactions. This review discusses the principles, applications, and advancements in SPR and surface plasmon resonance imaging (SPRI) in optical-based biosensors. SPR measures refractive index changes near thin metal layers in response to biomolecular interactions, enabling real-time, high-sensitivity measurements without labels. The technique is widely used in biomedical, proteomics, genomics, and bioengineering fields. Key applications include interaction analyses, conformational change studies, mutation detection, high-throughput screening, and cellular analysis. Signal amplification strategies, such as using metal nanoparticles, magnetic nanoparticles, and carbon-based nanomaterials, enhance SPR sensitivity. Recent advances in SPR technology, including improvements in spatial resolution and multiplexing capabilities, continue to expand its utility in various biological and clinical applications.