The aim of this study is a highly sensitive and selective label-free surface plasmon resonance (SPR) aptasensor preparation for the specific detection of human activated protein C (APC). In the first step, DNA aptamer was complexed with N-methacryloyl-L-cysteine (MAC) monomer. Then, cyanamide and 2-hydroxyethyl methacrylate solution was mixed with the DNA-Apt/MAC complex. Two different SPR sensors (Random-DNA and HEMA-MAC polymeric films) were also prepared by following the same experimental procedure. The characterization of SPR aptasensors was done by contact angle, atomic force microscopy, and ellipsometer analysis. Selectivity studies of SPR aptasensors were performed in the presence of bovine serum albumin, hemoglobin and myoglobin. Desorption studies were performed by using 0.025 M NaCl solution. The limit of detection (LOD) and limit of quantification (LOQ) values of DNA-Apt SPR aptasensor was determined as 1.5 ng/mL and 5.2 ng/mL.