Cold stress is an important environmental stressor in aquaculture, yet the integrated biochemical and physiological responses of fish to acute low-temperature exposure remain insufficiently investigated. In this study, we investigated the effects of acute cold stress on Luciobarbus capito using an integrated approach combining biochemical assays, gut microbiota profiling, and liver metabolomics. Fish were exposed to 12°C for 96 h, with a 22°C group serving as the control. Cold stress induced marked time-dependent physiological disturbances. Hepatic T-SOD and CAT activities were significantly reduced, whereas MDA content increased, indicating oxidative imbalance in the liver. Serum ALT and AST activities increased after 24 h, while ACP activity declined from 12 h onward, suggesting tissue injury under cold exposure. In parallel, cold stress significantly altered the intestinal microbial community, as reflected by reduced microbial richness, depletion of potentially beneficial taxa such as Cetobacterium, and enrichment of stress-associated taxa such as Pseudomonas. Liver metabolomic profiling identified 172 differential metabolites and revealed substantial alterations in pathways related to glycerophospholipid metabolism, α-linolenic acid metabolism, pantothenate and CoA biosynthesis, and ascorbate and aldarate metabolism. These metabolic alterations were associated with changes in membrane lipid metabolism, energy-related pathways, and antioxidant status under cold stress. Correlation analysis further revealed significant associations between altered intestinal bacterial genera and hepatic differential metabolites, suggesting coordinated intestinal and hepatic responses to acute cold exposure. Overall, these findings indicate that acute cold stress induces oxidative imbalance, intestinal microbial restructuring, and hepatic metabolic disturbance in L. capito, providing an integrated view of the biochemical and physiological responses of fish to low-temperature stress.