Abstract: Tetracycline is a tetracycline antibiotic. Tetracycline residues in the environment have chronic effects on aquatic organisms. However, there were no aquatic life criteria for tetracycline in fresh water in China. The acute and chronic toxicity data of tetracycline on freshwater aquatic organisms in China were collected and screened. A total of twelve acute toxicity data from seven phyla and twelve families and nine chronic toxicity data from seven phyla and nine families were obtained. The aquatic life criteria of tetracycline were derived by toxicity percentile rank and species sensitivity distribution methods. Finally, the short-term and long-term water quality criteria of tetracycline were 61.650 and 9.439 μg/L, respectively, derived by the species sensitivity distribution method. The ecological risk of tetracycline in some water bodies in China was assessed by the entropy method and the safety threshold method, finally the entropy method was used to evaluate the ecological risk level of tetracycline in freshwater environment in China. The results of the assessment suggested that the areas surveyed with ecological risk exposed to tetracycline mainly located in the Nanming River of Guiyang Province, and most other areas were basically risk-free. The research conclusions could provide scientific basis for formulating the water quality standards in terms of tetracycline, protecting freshwater aquatic life and managing water eco-environment.