Abstract: To investigate the residue risks of avermectin to aquatic organisms, a trace detection method for avermectin in aquaculture water was established using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Zebrafish (Danio rerio) were used as a model organism for gradient toxicity tests (0.0335-0.0833 mg a.i./L). A static exposure method was employed to observe behavioral changes, mortality, and related physiological indicators in zebrafish, as well as differences in gut microbiota structure and function. The results showed that the 96-hour LC₅₀ value of avermectin for zebrafish was 0.0680 mg a.i./L, which was classified as highly toxic substance. At non-lethal exposure concentrations, the locomotor activity of zebrafish was significantly reduced. The content of malondialdehyde (MDA) and the activity of catalase (CAT) in zebrafish increased with increasing exposure concentrations, while the activity of superoxide dismutase (SOD) showed a promoting effect at low concentrations and an inhibitory effect at high concentrations. Additionally, the exposure to avermectin significantly reduced the diversity and evenness of the gut microbiota in zebrafish, altered the compositional structure of the microbiota, and particularly increased the abundance of conditional pathogens. Functional prediction of the microbiota indicated that this would lead to dysregulation of normal metabolism and immune functions. These findings suggest that avermectin can produce long-term toxic effects on zebrafish through stress-induced oxidative damage and disruption of gut microbiota composition and function. The residue issues of avermectin in aquaculture should be highly concerned to ensure the safety and quality of aquatic products.