Cold chain disruption during logistics is a critical cause of food quality degradation. This study simulated 3 possible cold chain patterns and combined liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-MS (GC-MS) to explore the quality deterioration and metabolic changes of fresh beef under cold chain disruption. The findings revealed that the cold chain interruption led to an upregulation of metabolites associated with lipid metabolism and protein degradation, which are linked to severe quality deterioration. Specifically, L-valine, L-isoleucine, β-alanine, 12-ketoeicosatetraenoic acid, and 5,6-epoxyeicosatrienoic acid exhibited upregulation. Tricarboxylic acid cycle intermediate metabolites oxoglutaric acid is a pivotal up-regulated metabolite that links protein degradation processes and post-slaughter energy production. Additionally, carbonyl compounds showed significant alterations in the beef under cold chain disruptions. Moreover, the later stage of cold chain disruption triggered a rapid collapse of beef quality. This study combined GC-MS and LC-MS to identify key metabolites associated with quality deterioration caused by cold chain disruption, providing a new perspective on beef quality monitor in cold logistics circulation.