Abstract: Immunomodulatory peptide was prepared from Thunnus albacores meat assisted by ultrasonic wave. Trypsin was found to be the most suitable protease based on the relative proliferation rate of mouse macrophages. The hydrolysis conditions were determined through response surface optimization experiments. Results showed that, the optimal parameters were as follows: 5 h of enzymolysis time, 37.6 ℃ of temperature, and 11 min of ultrasonic time, where the relative proliferation rate of macrophages was 40.04%±2.03%. The amino acid composition and molecular weight of the peptide were also analyzed. The results showed that the essential amino acid content was 41.42%±1.24%, the hydrophobic amino acid content was 26.49%±0.79%, and the molecular weight was concentrated in 4.7~6.5 kDa. The research results would provide the theoretical and technical support for high value utilization of yellowfin tuna.