Abstract: In order to screen for meat tenderization treatment methods targeting a large number of eliminated laying hens in poultry farms, the ultrasound combined with calcium chloride and biological enzyme methods were used to tenderize chicken breasts. The shear force was chosen as the key indicator, and the optimal ultrasound parameters were selected. The texture, water retention, protein distribution, total thiol and amino acid contents as well as the microstructure changes were mainly analyzed. Results showed that the optimal ultrasound parameters were ultrasound of 240 W, frequency of 22 kHz, water bath temperature of 35 ℃, and treatment time of 10 minutes. Compared to the untreated group, the shear force of chicken breast meat significantly decreased by 64.02% after the ultrasound combined with protease treatment. The hardness, stickiness, and chewiness of chicken breast meat decreased, with a significant reduction of 56.54% in chewiness, which was significantly better than that of calcium chloride. Meanwhile, the degree of cooking loss and centrifugation loss of meat were also lower than that of calcium chloride. The electrophoresis results showed that the high molecular weight myosin heavy chain was degraded near 116 kDa, indicating that some myofibrillar proteins were decomposed. Among them, the total thiol content also continuously decreased, and the chicken proteins were oxidized. When the ultrasound power continued to increase to 300 W, the content of essential amino acids threonine, valine, and lysine in chicken breast significantly decreased. Microscopic observation showed that the apparent and cross-sectional structures of chicken breast underwent disintegration, deep muscle fiber separation, decomposition, and fracture. In summary, the tenderness and quality of chicken breast meat were significantly improved after ultrasound combined with protease treatment.