Abstract: In response to the problems of water loss, fat oxidation, and decreased freshness caused by prolonged thawing of meat, an efficient multi-frequency ultrasonic method was adopted for thawing. The effects of frequency combination on the qualities of beef brisket were studied using multi-frequency ultrasound at 22, 28 and 40 kHz for processing. This study explored the effect of multi-frequency combined ultrasound on the thawing quality of beef brisket from the perspectives of thawing time, water retention, color, freshness, texture characteristics, distribution of myofibrillar protein and muscle fiber structural features. Results showed that, the 28/40 kHz had the best effect on beef brisket in the ultrasonic thawing treatment. Compared with static water thawing way, the thawing time (524 s) and thawing loss rate (3.55%) of beef brisket under 28/40 kHz were significantly (P<0.05) reduced. Compared with fresh meat, the meat hardness (338.68 N), chewiness (456.81 mJ) and shear force (91.17 N) were significantly reduced (P<0.05). But there was no significant difference (P>0.05) in elasticity (4.09 mm), adhesive strength (206.20 N), cooking loss rate (33.11%), TVB-N (3.13 mg/100 g) and MDA (0.177 mg/kg) values compared to fresh meat, respectively. The combination of this frequency maximized the freshness of beef brisket and improved the tenderness. SDS-PAGE results showed that the large molecule myosin heavy chain (200 kDa) degraded at this frequency combination. Microscopic observation also showed that the cross-section of beef brisket muscle fibers thawed at this frequency combination was relatively flat, and the degree of damage to muscle fiber bundles was minimal. 28/40 kHz dual frequency sequential ultrasound treatment could effectively improve the thawing quality of frozen beef brisket.