The preparation and performance of an amperometric immunosensor，based on the use of layer-by-layer self-assembly technique，for the specific detection of prostate specific antigen(PSA) was reported. The immunosensor was developed by immobilizing the anti-PSA through layer-by-layer self-assembly of hyaluronic acid(HA)，thionine(Thi) and gold nanoparticles(nano-Au) on chitosan(Cs) modified glassy carbon electrode(GCE).The bovine serum albumin(BSA) was employed to block the possible remaining active sites of the nano-Au monolayer，avoiding the non-specific adsorption.The electrochemical characteristics of the immunosensor were studied by cyclic voltammetry(CV) and differential pulse voltammetry(DPV).The factors influencing the performance of the immunosensor were studied in detail.Under the optimized conditions，the linear ranges of the immunosensor were from 0.5 μg/L to 4 μg/L and from 4 μg/L to 25 μg/L with correlation coefficients more than 0.993，and a detection limit of 0.2 μg/L(estimated to be 3× the standard deviation of zero-dose response).The practical utilization of the immunosensor has been demonstrated through the determination of PSA in human serum samples.The results determined with the proposed immunosensor agreed fairly well with those obtained by the enzyme linked immune-sorbent assay(ELISA) technique.