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 with a low limit of detection of 0.8 pg mL. 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.