An immunosensing system based on surface plasmon resonance (SPR) was used for on-line detection and characterization of carbohydrate molecules separated by high-performance liquid chromatography. These analytes, with or without serum, were continuously separated and analyzed in the combined liquid chromatography-surface plasmon resonance (LC-SPR) system. By using weak and readily reversible monoclonal antibodies, the SPR system allowed specific on-line monitoring of the substances. To increase the specificity of the immunosensor, nonrelevant antibodies were used as reference in a serial flow cell. The sensitivity of the LC-SPR system was dependent on molecular weight of the carbohydrate, affinity of binding, and design of the sensor.