The ubiquitous presence of antibiotic residues in aqueous environments poses a great potential threat to aquatic organisms. Nevertheless, the behavioral effects of environmentally realistic levels of antibiotics remain poorly understood in fish species. In this study, the behavioral impacts of enrofloxacin, one of typical fluoroquinolone antibiotics that is frequently detected in aquatic environments, were evaluated by the classic light-dark test (LDT) and novel tank task (NTT) in zebrafish. Furthermore, the effects of enrofloxacin exposure on the microbiota-gut-brain axis were also assessed to reveal potential affecting mechanisms underlying the behavioral abnormality observed. Our results demonstrated that zebrafish exposed to 60 μg/L enrofloxacin for 28 days took significantly longer to enter the stressful area of the testing tank and spent significantly less time there in both the LDT and NTT, indicating abnormal anxiety-like behaviors induced by the exposure. In addition, exposure to enrofloxacin at 6 and 60 μg/L resulted in a significant elevation in Bacteroidetes and a marked decline in the Firmicutes/Bacteroidetes ratio of the gut microbiota. Moreover, the intestinal contents of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), glucagon-like peptide 1 (GLP-1), and 5-hydroxytryptamine (5-HT) in zebrafish were significantly upregulated, whereas those of plasma adrenocorticotropic hormone (ACTH) and cortisol (COR) were markedly downregulated upon enrofloxacin exposure. Incubation of zebrafish with a high dose of enrofloxacin (60 μg/L) also resulted in evident increases in the contents of corticotropin-releasing hormone (CRH), brain-derived neurotrophic factor (BDNF), and neuropeptide Y (NPY) in the brain. Fortunately, no significant alteration in the expression of glial fibrillary acidic protein (GFAP) was detected in the brain after enrofloxacin exposure. Our findings suggest that the disruption of the microbiota-gut-brain axis may account for enrofloxacin-induced anxiety-like behaviors in zebrafish. Since the disruption of microbiota-gut-brain axis may give rise to various clinical symptoms, the health risk of antibiotic exposure deserves more attention.