In this study, Concanavalin A (Con A) imprinted surface plasmon resonance (SPR) biosensor was prepared for the determination of Con A. Con A imprinted SPR biosensors were characterized by atomic force microscopy, ellipsometer, and contact angle measurements. The kinetic studies were performed with pH scanning. For this purpose, Con A solutions were prepared with different pH buffers and were used for the detection of Con A. The kinetic analysis of the SPR biosensor was with Con A solutions ranging from 0.01 to 50 ng/mL. The limit of detection and limit of quantification were calculated at 0.0011 and 0.0038 ng/mL, respectively. Selectivity studies were conducted using SPR biosensors that were either Con A imprinted or non-imprinted, as well as competing molecules such as bovine serum albumin and myoglobin, which have a similar molecular structure. In order to determine Con A in clinical studies, kinetic analyzes were performed with Con A solutions prepared in artificial plasma and Jack bean solutions. The recovery of Con A was calculated using the obtained sensorgram data. Four consecutive analyses were performed on the same day to test the Con A molecule’s reusability. In order to test the reusability of the biosensor at various points in time, kinetic analyses were also performed during the first, second, fourth, and sixth months.