Abstract: By coupling molecular imprinting, chitosan biosorption and TiO2 photocatalysis, selenium nanoparticles (Se NPs) were self-assembled in a controlled manner on the molecular imprinting sites of zeolite-chitosan-TiO2 microspheres. Se NPs with different size ranges and areal densities were individually synthesized by controlling the rapid adsorption of molecular-imprinted nanocomposites and photocatalytic reaction of TiO2 nanoparticles. In order to improve the sensitivity and specificity of rapid diagnostic detection, Se NPs were self-assembled again into high-order and spherical stable structures with an average size of 80 nm by well-defined monomer units, after separation form zeolite-chitosan-TiO2 microspheres with a stabilizer of 0.3% (v/v) bovine serum albumin. Due to the biological activity, spherical-shaped Se NPs were used for dot-blot immunoassays with multiple native antigens for rapid serodiagnosis of human lung cancer. The sensitivity of the dot immunoassays for detecting progastrin-releasing peptide (ProGRP) was 75 pg/mL. The detection time of colloidal Se dot immunoassays for ProGRP was only 5 min. No positive results were observed with other commonly potential interfering substances, including carcinoembryonic antigen, α-fetoprotein antigen and BSA. The research presents a simple and green method for the reuse of SeO32- and controlled synthesis of Se NPs for biological and medical applications by bioaffinity adsorption and photoreduction