In this study, we have developed an electrochemical immunosensor for the detection of prostate-specific antigen (PSA) based on gold nanoparticles (AuNPs) and a chitosan (CHI) nanocomposite film coated on a screen printed electrode (SPE). The modified SPE was characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immunosensor was fabricated by sequential immobilization of anti-PSA primary antibody, PSA antigen and HRP-tagged secondary antibody on the surface of AuNPs/CHI/SPE. When this immunosensor was placed in 2.5 mM H2O2, as HRP substrate, and 1 mM methylene blue (MB), as redox mediator, greatly amplified immunosensing signals were observed. Immunosensing signals for the reduction of H2O2 were monitored in phosphate buffer (pH 7.0) by CV and square wave voltammetry (SWV). Under optimized conditions steady state current increased linearly with PSA concentration over the range 1-18 ng/ml with a detection limit of 0.001 ng/ml. The fabricated immunosensor demonstrated excellent sensitivity, stability and reproducibility and therefore was successfully applied to analyse PSA in biological samples. Finally, the results indicate that the proposed immunosensor has potential in clinical screening of cancer biomarkers.