Abstract: Objective To prepare time-resolved fluoroimmunoassay (TRFIA) kit for detecting human alpha-fetoprotein (hAFP).Methods Sandwich TRFIA kit for hAFP detection was developed using monoclonal antibody of anti-AFP.Results AFP-TRFIA kit was capable of detecting AFP within the range of 1-1 000 U/ml with sensitivity of 0.17 U/ml and without cross-reactivity with CEA, CA12-5, CA15-3, or CA19-9. The intra- and inter-assay coefficients of variation were (3.3-5.9)% and (3.7-6.5)%, respectively. The prepared AFP-TRFIA reagent could withstand preservation at 4℃ for 1 year and at 37℃ for 7 days with the cutoff value of 12U/ml in healthy subjects (n=426). The correlation coefficient of the detection results between this kit and commercially available AFP kit (Wallac OY, Finland) for 60 blood samples was 0.995.Conclusion The prepared TRFIA kit for hAFP detection meets the demand of clinical application with good sensitivity, precision, specificity, stability and accuracy.