A new near-infrared-based photothermal immunosensing strategy was developed for the sensitive and feasible detection of human chorionic gonadotropin (HCG) by use of a Prussian blue nanoparticle-based photothermal conversion system. Prussian blue nanospheres synthesized by the one-pot method were used for the labeling of anti-HCG detection antibody. A sandwich-type immunoreaction was initially conducted on a monoclonal anti-HCG antibody-coated microplate with a nanoparticle-labeled signal antibody. Accompanying formation of the sandwiched immunocomplex, Prussian blue nanospheres caused photothermal conversion under 980-nm laser irradiation, thereby resulting in an increase of the temperature of the detection system measured by a portable digital thermometer. The properties and factors influencing the analytical performance of the photothermal immunoassay were studied in detail. Under the optimal conditions, the Prussian blue nanoparticle-based photothermal immunoassay exhibited good temperature responses relative to target HCG concentrations within the dynamic range of 0.01-100 ng mL at a low detection limit of 5.8 pg mL. This system also displayed good anti-interference behavior with regard to other cancer biomarkers, good reproducibility, and relatively long storage stability. The method accuracy was evaluated for analysis of human serum specimens, giving results that matched well with those obtained with a commercial HCG enzyme-linked immunosorbent assay kit. Importantly, this protocol is promising for advanced development of photothermal immunoassays. Graphical abstract.