A facile and highly sensitive determination of prostate-specific antigen (PSA) is of great significance for the early diagnosis, monitoring and prognosis of prostate cancer. In this work, a disposable and label-free electrochemical immunosensing platform was demonstrated based on chitosan-graphene-modified indium tin oxide (ITO) electrode, which enables sensitive amperometric determination of PSA. Chitosan (CS) modified reduced graphene oxide (rGO) nanocomposite (CS-rGO) was easily synthesized by the chemical reduction of graphene oxide (GO) using CS as a dispersant and biofunctionalizing agent. When CS-rGO was modified on the patterned ITO, CS offered high biocompatibility and reactive groups for the immobilization of recognition antibodies and rGO acted as a transduction element and enhancer to improve the electronic conductivity and stability of the CS-rGO composite film. The affinity-based biosensing interface was constructed by covalent immobilization of a specific polyclonal anti-PSA antibody (Ab) on the amino-enriched electrode surface via a facile glutaraldehyde (GA) cross-linking method, which was followed by the use of bovine serum albumin to block the non-specific sites. The immunosensor allowed the detection of PSA in a wide range from 1 to 5 ng mL(-1) with a low limit of detection of 0.8 pg mL(-1). This sensor also exhibited high selectivity, reproducibility, and good storage stability. The application of the prepared immunosensor was successfully validated by measuring PSA in spiked human serum samples.