Katsuwonus pelamis processing by-products, including viscera, fish head, fish gills and fish skin, contain protein, fat and other nutrients. Ineffective utilization of these by-products will lead to the waste of nutritious resources and serious environmental pollution. In order to provide a practical method to improve the utilization of by-products, the peptone was prepared by ultrasonic-assisted enzymatic hydrolysis of K. pelamis by-products by trypsin. The effects of time, temperature and power of ultrasonic on the changes of protease activity, degree of hydrolysis, nitrogen recovery, TCA-soluble nitrogen recovery and molecular weight distribution of the hydrolysate were investigated during the ultrasonic-assisted enzymatic hydrolysis of K. pelamis by-products. The conditions of peptone preparation were optimized by single factor experiment. The results showed that the enzymatic activity of trypsin was significantly changed by ultrasound treatment, and the activity of trypsin increased within 30 min under the ultrasonic power 120 W. However, after 30 min, the activity of trypsin decreased obviously, until 4 h later the enzyme activity tended to decrease gently. The power of ultrasonic and the temperature of enzymatic hydrolysis all affect the enzymatic efficiency of enzymatic hydrolysis of K. pelamis by-products. Under the conditions of ultrasonic power 240 W and 50 ℃, the recovery of nitrogen increased by 18.66%, the degree of hydrolysis increased by 30.43% and the recovery of TCA-soluble nitrogen increased by 33.20%, compared with non-ultrasound group. And ultrasound also can effectively improve the small molecule peptide in the enzymatic solution. The optimum conditions were temperature 55 ℃, ultrasonic power 240 W, hydrolytic time 4 h. Under these conditions, the recovery of nitrogen was 81.40% and the recovery of TCA-soluble nitrogen was 40.75%.