Abstract: Bovine blood is one of the main by-products of bovine slaughtering and processing. The preparation and antioxidant activity of bovine blood peptides has been investigated systematically to promote their high-end value in industrial utilization. Taking the bovine hemoglobin (BH) as the raw material, this study aims to prepare the BH antioxidant peptide crude product using two-step hydrolysis. The reference indexes were taken as the hydrolysis degree and the ABTS·free radical scavenging ability. Six proteases were also employed for the hydrolysis to identify and select the optimal protease. Specifically, a systematic evaluation was made to investigate the effect of different solid-liquid ratios, enzyme concentration, reaction temperature, pH value, and reaction time on the initial hydrolysate, including antioxidant activity using ABTS·free radical scavenging. The remaining five proteases were used to further hydrolyze the first BH hydrolysate, where the optimal protease was selected for the single-factor test. Furthermore, the influencing parameters were assessed during enzymolysis on the antioxidant capacity of the bovine hemoglobin second hydrolysate. The results showed that: 1) The best effect was found in the first enzymatic hydrolysis of flavorzyme among the six proteases. The highest hydrolysis degree and the ABTS·scavenging ability were also observed in the first BH hydrolysate (P<0.05); 2) The BH was hydrolyzed at pH value is 7.5 and 50 ℃ for 120 min, when the solid-liquid ratio and flavorzyme content were 40 g/L and 4 000 U/g, respectively, indicating the highest antioxidant capacity of the first hydrolysate; 3) The alcalase demonstrated the best second enzymatic hydrolysis effect; 4) The BH first hydrolysate was hydrolyzed (pH values are 9.0; 40 ℃; 60 min), when the concentration of alcalase was 3 000 U/g. While the hydrolysis degree (46.06%±0.08%) and ABTS free radical scavenging ability (314.15±1.09) μmol TE/g protein) of the second hydrolysate were significantly higher than other protease groups (P<0.05). The single-factor test show that the enzyme concentration and reaction time were further optimized using the response surface method (RSM), with the ABTS free radical scavenging ability as the response value. Furthermore, the regression equation was obtained after RSM optimization, indicating the enzyme concentration and reaction time on the antioxidant capacity of the BH hydrolysate. The optimal parameters were achieved to prepare the BH antioxidant peptide crude product with the solid-liquid ratio of 40 g/L. The first enzymatic hydrolysis included: Amount of flavorzyme is 3 800 U/g, enzymatic hydrolysis time is 130 min, temperature is 50 ℃, and pH value is 7.5, while the second enzymatic hydrolysis process included: Amount of alcalase is 2 900 U/g, time is 60 min, temperature is 40℃, and pH value is 9.0. The ABTS free radical scavenging ability was achieved at (333.62±6.29) μmol TE/g protein, indicating excellent scavenging capacity and antioxidant activity. A systemic and effective procedure was obtained to extract the food-derived antioxidant peptides. The finding can provide new insight into the comprehensive processing and utilization of by-products, not limited to the bovine blood. A theoretical basis was also offered for the research and development of food-derived antioxidant peptides.