Eu-chelate were used to construct a two-site sandwich-type assay for pepsinogen I (PGI) with time-resolved fluoroimmunoassay (TRFIA) as a detection technique. On the noncompetitive assay, captured monoclonal antibodies (McAbs) coated on wells were directed against a specific antigenic site on the PGI. Another McAbs, called as labeling McAbs, were prepared with the Eu-chelate of N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N,N,N-tetraacetic acid and directed against a different antigenic site on the PGI. The fluorescence counts of bound Eu 3+-McAbs were measured with the auto DELFIA_ 1235 system. The PGI in sera from healthy volunteers were determined by PGI-TRFIA. The within-run and between-run CVs of the PGI-TRFIA were 1.9% and 4.7%, respectively, and the recovery rate was 102.65%. The assay had a detection limit of 0.05 μg·L -1. The PGI-TRFIA provided a linear response from 3.5 to 328 μg·L -1. The cross-reacting rate with pepsinogen II was negligible. The linear correlation of PGI-TRFIA and radioimmunassay measurements resulted in a correlation coefficient of 0.977. The means of healthy volunteers were 154±43 μg·L -1 for serum PGI. The availability of a highly sensitive, reliable, and convenient method for quantifying PGI will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastritis and severe atrophic gastritis. Eu-chelate were used to construct a two-site sandwich-type assay for pepsinogen I (PGI) with time-resolved fluoroimmunoassay (TRFIA) as a detection technique. On the noncompetitive assay, McAbs coated on wells were directed Another McAbs, called as labeling McAbs, were prepared with the Eu-chelate of N- (p-isothiocyanatobenzyl) -diethylenetriamine-N, N, N, N-tetraacetic acid and directed against a different antigenic site on the PGI. The fluorescence counts of bound Eu 3 + -McAbs were measured with the auto DELFIA 1235 system. The PGI in sera from healthy volunteers were determined by PGI-TRFIA. The within-run and between-run CVs of the The PGI-TRFIA was 1.9% and 4.7% respectively, and the recovery rate was 102.65%. The assay had a detection limit of 0.05 μg · L -1. The PGI-TRFIA provided a linear response from 3.5 to 328 μg · L -1. 1. The cross-reacting rate with pepsinogen II was negligible. The linear correlation of PGI-TRFIA and radioimmunassay measurements resulted in a correlation coefficient of 0.977. The means of healthy volunteers were 154 ± 43 μg · L -1 for serum PGI. The availability of a highly sensitive, reliable, and convenient method for quantifying PGI will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastritis and severe atrophic gastritis.