Abstract：Objective To establish time-resolved fluoroimmunoassay for IgG antibody of human cytomegalovirus（HCMV）. Methods The HCMV pp150+pp52+pp65 antiage was coated on microplates, the goat anti-human IgG was labeled by Europrum. The enhancement solution was β-naphthalene thrifluoroacetone. The 1020 samples were analyzed by self-made kits and commercial kits. Results The detection limit, repeatability, compliance rate of negative and positive national standard of self-made time-resolved fluoroimmunoassay kit was in line with national accreditation standards. The linear range of time-resolved fluoroimmunoassay was from 2.0 to 256.0 RU/mL, the linear correlation coefficient was up to 0.99. The analysis performance of kits had no significant changes after storing at 37℃ for six days. The testing results of self-made kit with reference kit for 1020 samples was linear correlation (Y = 0.9594X +0.3252, r = 0.9918). Conclusion The time-resolved fluoroimmunoassay for IgG antibody of human cytomegalovirus established in this study and self-made kit have high sensitivity, specificity, good precision and high accuracy. It could meet clinical demand for the analysis of IgG antibody of human cytomegalovirus.