Abstract: In order to achieve a high-value utilization of pigeon blood, a by-product during the processing of meat pigeon, this study focused on the preparation of antioxidant and anti-aging peptides from pigeon blood through enzymatic hydrolysis. First, pretreatments of pigeon blood were adopted to help the hemoglobin in blood erythrocytes dissolved out. Then, different proteases were employed to enzymatically hydrolyze the hemoglobin in pigeon blood, and the DPPH and ABTS⁺ radical scavenging activity of the resultant hydrolysate, as well as the tyrosine kinase inhibitory activity were selected as screening indexes to optimize the enzymolysis conditions. The enzymatic hydrolysis process of active peptides in pigeon blood was optimized by using single-factor and orthogonal experiments. Result showed that ultrasonic treatment for 5 min at 100 W owned the highest dissolving hemoglobin content for 192.65 mg/mL, which indicating the best hemolysis effect. The DPPH and ABTS⁺ radical scavenging activity and tyrosine kinase inhibitory activity of hydrolysate obtained from pepsin treatment were all basically higher than those from other proteases, and the protein recovery was also relative higher, revealing pepsin as the most effective protease for the enzymatic hydrolysis. The optimum conditions for the preparation of antioxidant and anti-aging peptides from pigeon blood established: blood-to-water ratio at 1:1.2, protease addition at 12000 U/g, hydrolysis time at 4 h, hydrolysis temperature at 37 ℃, and pH at 2.0. Under this process, the tyrosine kinase inhibition rate of the obtained pigeon blood hydrolysate reached the highest, at 45.93%, and the ABTS⁺ scavenging rate was 83.84%. The hydrolysate was mainly consisted of peptides with molecular weight smaller than 5000 Da, accounting for 78.98% of the total. And the essential amino acids, hydrophobic amino acids, and antioxidant amino acids accounted for 42.01%, 38.62%, and 27.22% of the total amino acids, respectively, which indicated excellent nutritional value and antioxidant and anti-aging effects of the hydrolysate obtained from pigeon blood. All these provided a fundamental basis for the development of bioactive peptides from pigeon blood and the reutilization of meat pigeon processing by-products.