In this study, a collagen-gelatin-gold nanoparticles (AuNPs) matrix, a material widely used in biosensor applications, controlled release systems, tissue engineering and many other biotechnology fields, was applied to the apta-sensor technology. The matrix was characterized using techniques like scanning electron microscopy (SEM), thermal gravimetric analysis (TGA) and fourier-transform infrared spectroscopy (FTIR). Immobilization conditions were optimized through immobilization of thrombin binding aptamers (TBA) on to the collagen-gelatin-AuNPs surface. Polymer, cross-linker, AuNPs and aptamer concentrations in addition to pH were optimized in the developed aptasensor systems. In addition, re-usability and reproducibility were examined for validation purposes. Thrombin levels as low as 1.97 nM were detected using screen printed carbon electrodes as transducer and Electrochemical Impedance Spectroscopy (EIS) as the electrochemical method.