This study prepared chromium(Ⅲ)-chelating peptides derived from steam-exploded pea protein (SEPPs-Cr(Ⅲ)) and evaluated its biological activities. The preparation conditions were optimized by one-factor-at-a-time (OFAT) method and response surface methodology (RSM) using the chelation rate of chromium(Ⅲ) as the response variable. The structures of steam-exploded pea protein-derived peptides (SEPPs) and SEPPs-Cr(Ⅲ) were characterized by ultraviolet-visible (UV-Vis) absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, zeta potential, particle size, and scanning electron microscopy (SEM). Their antioxidant and hypoglycemic activities in vitro were evaluated. The results showed that the optimal preparation conditions were determined as: peptide/chromium(Ⅲ) volume ratio of 1:3.7, pH 4.6, temperature of 37.5℃, and reaction time of 70 min. Under these conditions, the chelation rate of chromium(Ⅲ) was (31.67±0.75)%. Compared with SEPPs, SEPPs-Cr(Ⅲ) exhibited an increased particle size, significantly reduced amounts of negative surface charge, increased β-sheet content, and decreased random coil content, showing a rough, folded, and compact granular aggregate morphology. Spectral analysis confirmed that chromium(Ⅲ) primarily bound to amino, carbonyl, and carboxyl groups in the SEPP peptide chain, forming stable chelate structures. In vitro bioactivity studies indicated that the half maximal inhibitory concentrations (IC₅₀) of SEPPs-Cr(Ⅲ) against 1,1-diphenyl-2-picrylhydrazylradical (DPPH) and hydroxyl radicals were 5.14 and 4.6 mg/mL, respectively, which were significantly lower than those of SEPPs (11.6 and 8.79 mg/mL). The IC₅₀ against α-amylase and α-glucosidase were also significantly lower than those of SEPPs (0.39 versus 3.14 mg/mL and 0.59 versus 9.18 mg/mL). Furthermore, SEPPs-Cr(Ⅲ) exhibited a significantly stronger reducing capability than SEPPs. The findings provide a theoretical basis for developing organic chromium(Ⅲ) supplements.