Abstract: The effects of 200 W (UC200), 300 W (UC300) and 400 W (UC400) ultrasound assisted vinasse-curing on the formation of characteristic flavor in vinasse-cured beef were studied using beef as the fresh raw materials. The sensory quality, structure and morphology of myofibrillar proteins, fingerprint and odor-active values of volatile compounds were investigated to reveal the changes of ultrasound assisted vinasse-curing on the conformation of myofibrillar proteins and characteristic flavor in vinasse-cured beef. The correlation analysis was further employed to identify the correlation between the changes of myofibrillar proteins and the formation of characteristic flavor in vinasse-cured beef. The results demonstrated that 200 W (UC200), 300 W (UC300) and 400 W (UC400) ultrasound assisted vinasse-curing were able to improve flavor quality of vinasse-cured beef. Specifically, the ester aroma scores in the UC200, UC300, and UC400 groups increased from 0.50 in the control group (CK) and 6.25 in the non-ultrasonic fermentation group (NUC) to 7.23, 8.00, and 7.02, respectively (P<0.05), with the UC300 group showing a significantly higher score than the UC200 and UC400 groups. Conformation analysis revealed that, the UC300 group exhibited the highest intrinsic fluorescence intensity and surface hydrophobicity of myofibrillar proteins compared to other groups, reaching 2293.55 and 22.54 μg, respectively. Meanwhile, its α-helix content, average particle size, and surface roughness were the lowest, at 24.91%, 1.64 μm, and 0.43 nm, respectively. SPME-GC-MS results showed that a total of 43 volatile compounds were identified in the vinasse-cured beef. Esters and alcohols accounted for 71.15% of the total flavor compound content. The ester compounds content of UC300 group increased from 50.18 mg/kg of NUC group to 58.53 mg/kg (P<0.05). Partial least squares discriminant analysis and odor-active values indicated that ethyl caprylate, ethyl decanoate, phenethyl acetate, phenylacetaldehyde, nonanal, ethyl valerate, ethyl butyrate, ethyl benzoate, ethyl nonanoate, and ethyl phenylacetate were the key odor-active substances responsible for the increased characteristic flavor in the UC300 group. Spearman correlation analysis indicated that key odor-active compounds such as ethyl valerate and ethyl benzoate were negatively correlated with the α-helix of myofibrillar proteins, and positively correlated with the ester and alcohol aroma. Therefore, this research can provide critical references for the application of ultrasound in improving the aroma quality of vinasse-cured meat products.