Surface plasmon resonance (SPR) biosensors belong to label-free optical biosensing technologies. The SPR method is based on optical measurement of refractive index changes associated with the binding of analyte molecules in a sample to biorecognize molecules immobilized on the SPR sensor. Since late 1990's, SPR biosensors have become the main tool for the study of biomolecular interactions both in life science and pharmaceutical research. In addition, they have been increasingly applied in the detection of chemical and biological substances in important areas such as medical diagnostics, environmental monitoring, food safety and security. This chapter reviews the main principles of SPR biosensor technology and discusses applications of this technology for rapid, sensitive and specific detection of chemical and biological analytes.