In this study, fresh fly maggots were used as raw materials, two kinds of proteases were used to prepare the protein hydrolysates, and the in vitro antioxidant activity of hydrolysates were investigated. The molecular weight distribution of peptides of hydrolysates were detected by gel filtration chromatography, and the amino acid composition and content were determined by automatic amino acid analyzer. Finally, the small peptide with molecular weights of less than 3 000 Da were preliminarily separated by ultrafiltration membranes and the antioxidant activities of the peptides were determined. The results showed that neutral protease hydrolysate and alcalase hydrolysate obtained by fly maggots both had good free radical scavenging activity and Fe²⁺ chelating ability. The fractions with molecular weights below 3 000 Da reached more than 97% for both hydrolysates.The hydrolysate mainly contained 17 amino acids, and the three classes of amino acids (hydrophobic amino acid, acidic amino acid, basic amino acid) associated with antioxidants in the neutral protease hydrolysate and alcalase hydrolysate were accounted for 83.48% and 83.84%, respectively. The antioxidant activities of small peptides with molecular weights less than 3 000 Da obtained after preliminary separation by ultrafiltration membranes were significantly improved, and the IC₅₀ of the small peptide with hydrolysates below 3 000 Da against DPPH radical, ABTS radical, hydroxyl radical, and Fe²⁺ chelating were 0.40 mg/mL and 0.26 mg/mL, 0.35 mg/mL and 0.33 mg/mL, 0.92 mg/mL and 1.00 mg/mL, 1.09 mg/mL and 1.75 mg/mL, respectively. In conclusion, both neutral protease hydrolysate and alcalase hydrolysate prepared from fly maggots has strong antioxidant capacity, rich amino acids and high nutrition, which can provide reserve resources and theoretical basis for the development and application of natural antioxidant medicines.