The development of surface plasmon resonance (SPR) based immunosensor for thrombin detection was aimed. For this purpose, 3,3' Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DSP):6-mercapto-1-hexanol (MCH) mixed self-assembled monolayers (mSAMs) were formed on gold surfaces for immobilization of anti-thrombin antibody. The performance of the immunosensor was determined against the target protein thrombin at various concentrations using flow cell coupled SPR. The linear detection range of the immunosensor was 30.0-100.0 nM with an R-2 value of 0,992. Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined to be 6.0 nM and 30.0 nM, respectively. The selectivity of the immunosensor was tested against a non-target model protein, human serum albumin (HSA) and the obtained Delta RU value was found to be below the Delta RU value corresponding to the LOQ concentration for thrombin. The immunosensor's capability to detect thrombin in diluted complex serum matrix was also tested and the obtained Delta RU value (159 +/- 16) was compared with ARU value obtained for thrombin detection in PBS solution (137 +/- 19). Based on the results, it was shown that DSP:MCH interface is a promising immobilization platform for binding biological recognition elements for the development of biosensors. (C) 2019 Elsevier B.V. All rights reserved.