Abstract: Oxytetracycline, as an antibiotic pollutant, has the characteristics of large usage and strong ecological toxicity, and its environmental threshold and risk have been widely concerned by the academic circle. Based on the collected acute toxicity data of 13 species from 10 families of 5 phyla and chronic toxicity data from 10 species from 9 families of 3 phyla, the short-term and long-term water quality criteria of oxytetracycline in China were 61.2 μg/L and 18.7 μg/L, respectively, by using the species sensitivity distribution method. The derived criteria maximum concentrations and criteria continuous concentration were 34.0 μg/L and 7.4 μg/L, respectively. In addition, from 2007 to 2022, 803 oxytetracycline concentration values ​​in seven watersheds including the Yangze River, Yellow River and Haihe River in China were collected, and the risk entropy method, probability method and joint probability curve method were used for ecological risk assessment. The toxicity risk of phytochemicals to freshwater organisms is generally at a low level. The ecological risk of chronic toxicity is the highest in the Haihe River Basin and the lowest in the Pearl River Basin. Some areas, such as the tributaries of the Ziya River in the Haihe River Basin, have higher ecological risks. The research conclusions can provide scientific basis for the formulation of oxytetracycline water quality standards and the management of surface water environment.