A simple electrochemical immunosensor for the detection of prostate-specific antigen (PSA) is developed using three-dimensional Au nanoparticles anchored on a cobalt boride nanowire array (AuNPs/CoB NWA) electrode. AuNPs/CoB NWAs are synthesized by a simple electrodeposition method, which is first explored for immunoassay of PSA. An ingenious and simple signal amplification strategy without using the second labeled antibody to improve the sensitivity of the immunosensor is proposed. AuNPs/CoB NWAs can enhance the performance of electrocatalytic reduction of Nile blue A (NBA) to amplify signals. NBA is electropolymerized on AuNPs/CoB NWAs, forming an NBA/AuNPs/CoB NWA electrode. PSA antibody (anti-PSA) can be successfully attached to the NBA/AuNPs/CoB NWA electrode by a Au−N bond with AuNPs and the amino group of NBA, which causes an obvious decrease in the catalytic current due to the insulated anti-PSA. The current signal decreases linearly with the increase in PSA concentration because of the formation of an antibody−antigen immunocomplex. Depending on the above principle, the immunosensor based on NBA/AuNPs/CoB NWAs can exactly detect PSA in human serum samples. The lower detection limit of the PSA immunosensor is 2.77 pg•mL −1 (S/N = 3), and the high sensitivity is 484 μA•(ng•mL −1 )•cm −2 . It possesses a finer linearity with the PSA concentrations of 10 pg•mL −1 to 50 ng• mL −1 . The reproducibility, durability, specificity, and recovery of the immunosensor are further explored. The results are in good agreement with the standard enzyme-linked immunosorbent assay, providing a high research value on clinical screening and monitoring.