Abstract: To realize the high-value utilization of sesame meal, this experiment employed sesame meal as the raw material to investigate the optimal fermentation process for preparing sesame rough peptide (SRP) via liquid-state mixed fermentation with Bacillus subtilis and Bacillus velezensis (1:1), as well as evaluate its in vitro antioxidant activity. Meanwhile, the amino acid composition and relative molecular weight of sesame meal before and after fermentation were analyzed. Ultrafiltration technology was adopted to fractionate SRP, and the in vitro antioxidant activity of different fractions was determined; the fraction with the optimal activity was selected for subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. After predicting potential active peptide sequences through virtual screening, molecular docking technology was applied to perform simulation analysis on the screened peptides, thereby identifying potential antioxidant peptides, whose in vitro activity was further verified using artificially synthesized peptides.The results demonstrated that the optimal process parameters for SRP preparation were as follows: inoculum size of 9%, fermentation temperature of 41 °C, and fermentation duration of 35 h. Under these conditions, the actual yield of SRP reached 24.98%, with a DPPH radical scavenging rate of 79.1%. In terms of amino acid composition of post-fermentation SRP, the content of hydrophobic amino acids increased significantly from 71.295 mg/g to 156.117 mg/g. Moreover, most of the post-fermentation products were small-molecule oligopeptides composed of 4~6 amino acid residues. Ultrafiltration results indicated that the fraction with molecular weight (MW)<3 kDa exhibited the superior in vitro antioxidant activity. At a concentration of 5 mg/mL, its antioxidant activity was characterized by a DPPH radical scavenging rate of 90.63%, an ABTS⁺ radical scavenging rate of 73.17%, a total reducing capacity of 0.806, and a Fe²⁺ chelation rate of 77.83%. Four antioxidant peptide sequences (FRAFDAEL, FDGF, DLFR, and FLVR) were obtained via virtual screening. These peptides could exert antioxidant effects through binding via hydrogen bonds and hydrophobic interactions. The determination of in vitro antioxidant activity of artificially synthesized peptides confirmed that all of them possessed high antioxidant activity. This study provides a theoretical basis for the development and application of SRP-based antioxidant peptide products.