Abstract: With the rapid development of the meat processing industry, the inefficient utilization of large quantities of livestock and poultry by-products has become a critical constraint on the sustainable development of the industry. Porcine hearts are rich in high-quality proteins and various bioactive components, making them a potential resource for functional peptides. In this study, porcine cardiac muscle proteins were used as raw materials, and sequential enzymolysis was performed using alkaline protease and bromelain. The enzymolysis conditions were optimized through single-factor experiments and response surface methodology (RSM). Subsequently, the enzymolyzed products were sequentially separated by ultrafiltration using ultrafiltration tubes with molecular weight cut-offs (MWCO) of 10, 3, and 1 kDa. Peptidomic technology was employed to identify peptides, and bioinformatics prediction was combined to screen for potential antioxidant peptides. Molecular docking technology was used to analyze the binding affinity of the screened peptides to the target protein Keap1, and their activities were verified through in vitro chemical antioxidant assays. The results showed that the optimal enzymolysis conditions were: Temperature of 52 ℃, time of 7.5 h, substrate concentration of 0.5 g/mL, and enzyme addition amount of 1600 U/g. Under these conditions, the degree of hydrolysis (DH) reached 45.17%±0.08%, and the DPPH radical scavenging rate was 84.61%±0.31%. After ultrafiltration separation, the fraction M1 with the strongest antioxidant activity was obtained, which exhibited DPPH and ABTS⁺ radical scavenging rates of 85.55%±0.55% and 42.77%±0.45%, respectively. Six potential antioxidant peptides (FS, FD, YCC, WEL, ELF, TGAW) were screened out. Molecular docking results indicated that all six peptides could competitively block the formation of the Keap1-Nrf2 complex and bind to Keap1 protein through hydrogen bonds and hydrophobic interactions. Their binding energies were −56.9119, −66.9561, −73.6879, −84.2556, −82.0112, and −92.1879 kcal/mol, respectively, all lower than that of the positive control TX6 (−39.7879 kcal/mol), a high-efficiency and specific small-molecule ligand for Keap1. The underlying mechanism involved the competitive binding of these peptides to the Kelch domain of Keap1, thereby disrupting the protein-protein interaction between Keap1 and Nrf2, and ultimately activating the Nrf2-mediated antioxidant stress pathway. In vitro antioxidant verification confirmed that WEL and TGAW, with the lowest binding energies, possessed strong free radical scavenging capabilities and could serve as potential bioactive peptides for preventing oxidative stress-related diseases. This study provides a theoretical basis and technical support for the high-value utilization of livestock and poultry by-products and the development of natural antioxidants.