Based on the solid phase/liquid deposition CdSe quantum dots (QDs) were synthesized using selenium and cadmium-salt as precursor at room temperature. The TEM graph showed that the average size of CdSe QDs was ca. 8-10 nm. The mercaptoethylic-acid stabilization’s CdSe QDs exhibited strong ultraviolet absorption at 350 and 380 nm and strong fluorescence emission at 481.6 nm，respectively. When conjugated with pepsin, fluorescence peak intensity of CdSe QDs decreased considerably as well as fluorescence peak shifted to 467.2 nm. Under optimal conditions, a concentration of 5-50mg•L-1 of pepsin could be determined on the basis of fluorescence decrease ratio of CdSe QDs, with a detection limit 3σ of 0.36 mg•L-1 (n=10). The proposed method was applied to detection of the concentration of pepsin in human gastric juice.