A novel fluorescent immunosensor was developed based on the use of CuS nanoparticles (CuS NPs) as labels for the highly sensitive detection of human prostate cancer biomarker prostate specific antigen (PSA). In the presence of CuS NPs, the non-fluorescent substrate o-phenylenediamine could be oxidized into the stable fluorescent product 2,3-diamiophenazine at physiological pH. Throughout the reaction, no other oxidizing agents (e.g. hydrogen peroxide) were needed. The relatively mild oxidation conditions made the immunoassay robust, reliable and facile. The proposed immunoassay exhibited high sensitivity and specificity for the detection of PSA. A linear relationship between the fluorescent signals and the concentration of PSA was obtained in the range of 0.5 pg mL⁻¹ to 50 ng mL⁻¹, with a detection limit of 0.1 pg mL⁻¹ (S/N = 3). The proposed fluorescent immunoassay can be used as a promising platform for the detection of a variety of other biomarkers.