A sensitive immunoassay method was developed for the detection of HBsAg. A chemiluminescence label was prepared based on the co-immobilization of a secondary antibody and luminol on gold nanoparticles. HBsAg was targeted by a primary antibody, immobilized in polystyrene wells and a secondary antibody, co-immobilized on luminol–gold nanoparticles. After the formation of the immune sandwich, the luminescence intensity was recorded in the presence of hydrogen peroxide as an oxidant agent and Au³⁺ as an efficient catalyst for luminol oxidation. HBsAg was detected in the linear concentration range from 0.12 to 30 ng mL⁻¹ and had the detection limit of 14 pg mL⁻¹. The proposed method has been successfully applied to determine the HBsAg in patient serums. This method has excellent precision, a good detection limit, a wide linear range, a low cost and is user friendly. With these features, the proposed method is suitable for measuring antigen in patient serums.