The quality differences and Raman spectroscopic features of beef, which was frozen for 0–11 months followed by being thawed and displayed for 0–21 days were investigated. Frozen storage did not significantly affect the color blooming ability, in which minimal change in the color parameter redness and yellowness values of thawed beef in the later stage of frozen storage occurred. As such, it was not able to distinguish between frozen-thawed and non-frozen-thawed meat directly in terms of color changes. The total viable count and the total volatile basic nitrogen content increased sharply in the frozen-thawed meat subjected to 11-month frozen storage. The thiobarbituric acid reactive substances value increased dramatically from 0.18 to 0.29 mg/kg during 11-month frozen storage. Raman spectroscopy data showed that the stability of the protein secondary structure of thawed beef became worse after 3 months of frozen storage, of which the relative content of α-helix and β-sheet decreased significantly to 21.46% and 28.94% during 11-month frozen storage, respectively. The findings suggest that quality deterioration occurred for the frozen-thawed beef, and Raman spectroscopy could be a potential method to distinguish unfrozen meat from frozen-thawed meat.