This study employed multi-omics analysis to systematically evaluate the toxic effects of intraperitoneal injection of MC-LR on GIFT tilapia. The results showed that 96 h post injection, serum levels of aspartate GOT, GPT, LYZ, T-AOC, and SOD significantly decreased (p < 0.05). In contrast, hepatic levels of CAT and MDA significantly increased. The 16S rDNA sequencing method revealed a significant reduction in the α diversity of the intestinal microbiota. At the phylum level, the relative abundances of Firmicutes and Bacteroidota significantly decreased; at the genus level, several genera, including Bacteroides and Pseudomonas, also exhibited significant changes. Functional prediction indicated that the affected pathways were primarily related to metabolism and disease. Additionally, targeted metabolomics analysis showed a significant decrease in the levels of several SCFAs, such as butyric acid. Correlation analysis further elucidated the complex interactions between the intestinal microbiota, biochemical indicators, and SCFA metabolism. Overall, the study demonstrated that MC-LR induced oxidative stress and liver damage and led to intestinal microbiota imbalance and metabolic dysfunction in GIFT tilapia.