Abstract: The use of antibiotic can influence gut microbiota and impair memory function either positively or negatively. In order to investigate the effects of long-term exposure to doxycycline at environmental concentration on anxiety behavior, learning and memory, cognitive flexibility and intestinal flora, we took zebrafish as the animal model, and used doxycycline at different environmental concentrations (0, 0.01, 0.1 and 1 μg/L) to expose adult zebrafish to water for 21 days. Behavioral responses and intestinal flora alterations were assessed using the new tank test, Y-maze, light and dark test and zebrafish behavior high-throughput monitoring system. The results showed as follows: in the new tank experiment, the average swimming speed of zebrafish in 0.01, 0.1 and 1 μg/L concentration groups decreased significantly compared with the control group. Additionally, higher doxycycline concentrations correlated with reduced maximum swimming speed and total swimming distance, with a pronounced difference observed in the 1 μg/L group. Maze experiments indicated a significant increase in the residence time of zebrafish in the exploration arm at the 0.01 μg/L concentration, contrasting with decreased times in the 0.1 and 1 μg/L groups. The turning angle exhibited a similar pattern, increasing in the 0.01 μg/L group but decreasing in the higher concentration groups. In the light and dark test, zebrafish demonstrated an increased residence time in the light zone with rising doxycycline concentration. Zebrafish behavioral high-throughput monitoring system experiments further confirmed a significant decrease in total swimming distance with increasing doxycycline concentration, particularly notable in the 1 μg/L group. In terms of intestinal flora analysis, based on 16S rRNA sequencing technology, 1454814 original valid sequences were obtained from 12 intestinal samples, and 1314404 high-quality sequences were obtained after screening for subsequent analysis. At the same time, DADA2 method was used to reduce noise and cluster with 100% similarity. It was found that the abundance of intestinal flora of zebrafish in each concentration group decreased, and the abundance of bacteria genera such as Bosteria, Paracococcus and Acinetobacter decreased, while genera like Corynebacterium and Romatococcus increased. The Shannon index, Simpson index and Chao1 index were significantly lower in the exposed group, indicating decreased diversity. In conclusion, long-term exposure to doxycycline in environmental concentration induced behavioral changes in zebrafish, resulting in anxiety-like behavior. The 0.01 μg/L concentration improved short-term learning and memory ability and cognitive flexibility. Furthermore, doxycycline exposure led to changes in the abundance and diversity of zebrafish intestinal flora.