This study delves into the pivotal industrial process of efficiently managing livestock waste. Specifically, the study concentrates on harnessing the potential of bovine blood through enzymatic hydrolysis to produce antioxidant peptides. The whole bovine blood sample, subjected to a 90°C heat treatment for 30 min, underwent hydrolysis utilizing various commercial enzymes, alcalase, neutrase, and papain. Through neutrase hydrolysis (BB-N), we identified optimized conditions crucial for achieving heightened antioxidant activities and 40% protein recovery. Ultrafiltration with a molecular weight cutoff of 3 kDa was employed to concentrate the BB-N peptide, demonstrating the highest antioxidant and protein yield. The gel electrophoresis profile confirmed the denaturation of key proteins like albumin, globulin, and fibrinogen before digestion, while the BB-N derived after digestion contained peptides below 16 kDa. Post-concentration, the permeation of UF-3 kDa underwent purification, and the peptide sequence was discerned using liquid chromatography with tandem mass spectrometry. The exploration identified nine novel peptides- IWAGK, VDLL, MTTPNK, MPLVR, KIII, LPQL, TVIL, DFPGLQ, and VEDVK. Notably, the IWAGK sequence emerged as the most potent antioxidant activity peptide. Subsequent in-silico gastrointestinal digestion predicted structural changes in these peptides. While IWAGK, VDLL, MPLVR, LPQL, TVIL, and DFPGLQ could be fragmented into bioactive dipeptides and tripeptides, MTTPNK, KIII, and VEDVK exhibited resistance, suggesting potential circulation through the bloodstream to reach the target organ. Consequently, our study explores the potential use of BB-N as a novel dietary ingredient with health benefits. In vivo studies are needed to validate and extend our findings.