Storage temperature influences meat color stability and quality. This study was performed to quality change-associated metabolites profiles using a nontargeted liquid chromatography-mass spectrometry (LC-MS/MS)-based method. Beef longissimus dorsi samples were purchased immediately after slaughter, and then stored at room temperature, 4 °C and 0 °C. Water holding capacity (WHC), moisture content and pH value of the muscle samples were detected. Muscle samples and quality control samples were then prepared for nontargeted LC-MS/MS system, followed by identification of distinct metabolites. Pearson correlation coefficients between metabolites and quality indexes were calculated. Storage reduced pH values of beef, and room temperature and 4 °C displayed the lowest pH value. Moisture content and WHC in beef muscles, especially WHC declined obviously during the first 24 hr. The significantly altered metabolites profiles in meat samples at 0, 3.5, and 7 days during 4 °C storage were identified using LC-MS/MS. Most metabolites showed linear changes during storage (0 to 7 days). Using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we found 1(α)-naphthol, urocanic acid, tyramine, guanine, histamine, picolinic acid, 4-hydroxybenzaldehyde, and hypoxanthine were increased, and 2-(S-glutathionyl)acetyl glutathione and glutathione were decreased in beef during 4 °C storage. Correlation analysis showed there were significantly correlations between metabolites and meat quality indexes (WHC, moisture content, and pH). In summary, 1(α)-naphthol, urocanic acid, tyramine, guanine, histamine, picolinic acid, 4-hydroxybenzaldehyde, and hypoxanthine, proved to be harmful to human body, accumulated gradually, especially after 3.5 days during storage at 4 °C. While the contents of beneficial substances, including 2-(S-glutathionyl)acetyl glutathione and glutathione, were decreased, which provided reference for the nutrition guidance of using beef meat.