A novel electrochemical immunosensor based on gold-hydroxyapatite nanocomposite-chitosan film has been developed for the detection of prostate-specific antigen. The large surface area of the gold-hydroxyapatite nanocomposite and the porous morphology of chitosan allowed high antibody loading without the loss of bioactivity. The stepwise modification of the electrode was characterized with cyclic voltammetry. Different parameters, including the pH of a supporting electrolyte, the size of the gold nanoparticles, and the antibody concentration were optimized. Under the optimal conditions, the electrochemical immunosensor reliably detected prostate-specific antigen at concentrations from 3.5 to 30 ng/mL with a detection threshold of 2.6 ng/mL. The prepared biosensor was used to assay prostate-specific antigen in human serum samples, and the results were in good agreement with those obtained by enzyme-linked immunosorbent assays. The reported immunosensor exhibits rapid detection, good reproducibility, high sensitivity, specificity, and stable performance. The proposed strategy can be extended for the development of other biosensors. (C) 2011 Elsevier Ltd. All rights reserved.