A great deal of attention has been paid to preparation of bioactive peptides from proteins through enzymatic hydrolysis. Enzymatic hydrolysates of diverse food proteins exert various biological activities.In the present study, we prepared and characterized Grifola frondosa protein hydrolysate (GFPH)selenium chelating complex. Moreover, we assessed the effects of four main independent variables of the chelation conditions and selenium-binding capacity using response surface methodology (RSM). According to the results of RSM, the optimal chelation conditions were as follows: reaction time of 90 min, reaction temperature of 45 ℃, pH of 9.0 and the ratio of hydrolysates to sodium selenite (v:v) of 6:4. The content of selenium reached 2 979.45 ± 8.77 μg/g under the optimal conditions. Results of Fourier transform infrared spectroscopy (FTIR) and ultraviolet-visible (UV) spectroscopy indicated the successful incorporation of selenium into GFPH. The analyses of hydroxyl radical scavenging ability and ferric ion reducing antioxidant power (FRAP) were used to evaluate the antioxidant abilities of the newly synthesized chelate. Results showed that the antioxidant activities of GFPH-Se were superior to GFPH. The results of cellular uptake of GFPH-Se chelate in Caco-2 cells suggested that GFPH-Se chelate could be used as a potential source of Se supplement. Taken together, our findings indicated that the by-product of Grifola frondosa was a promising source for peptide-Se bio-products, which could be used as fungus-based functional supplements for humans with Se deficiency.