A dual-label time-resolved fluoroimmunoassay was established for simultaneously detecting pepsinogen Ⅰ (PGⅠ) and pepsinogen Ⅱ (PG Ⅱ) in human serum. Two capture monoclonal antibodies, 8003^# of PGⅠ and 8101^# of PGⅡ, were co-coated in 96 microtitration wells. The counterpart tracer monoclonal antibodies, 8016^# of PGⅠ and 8102^# of PGⅡ, were labeled with Eu³⁺ and sm³⁺-chelates, respectively. The samples were assayed by one-step sandwich protocol with the time-resolved fluorometry. The measurement ranges of PGⅠ were 0.2～300.0 μg/L with the within-run and between-run precision was 5.2% and 8.1%, and that of PGⅡ were 0.05～55.0 μg/L with the within-run and between-run precision was 7.1% and 11.7%, respectively. The average recovery rates of PGⅠ and PGⅡ were 96.9% and 103.7%, respectively. The results obtained by the dual-label assay agreed well with those by enzyme-linked immunosorbent assays of PGⅠ and PGⅡ, whose correlation ratio were 0.9426 of PGⅠ and 0.9396 of PGⅡ, respectively. The means of 300 healthy volunteers were (157.3 ±51.0) μg/L for serum PGⅠ,(10.6 ± 5.9) μg/L for serum PGⅡ, and (14.8 ± 4.3) for the PGⅠ/PGⅡ ratio. The normal ranges of serum PGⅠ levels for healthy volunteers were 55.3～259.3 μg/L, those of serum PGⅡ levels were less than 23 μg/L, the PGⅠ/PGⅡ ratio was more than 6. The proposed dual-label TRFIA for simultaneous detection of PGⅠ and PGⅡ is a simple, sensitive, and rapid method. It could provide serology high-screening of the samples for gastric diseases and would allow investigations into the possible diagnostic value of analysis in various clinical condition.