Abstract: The extensive application of tetracycline antibiotics in stock farming has brought potential hazards to people and animals. A well-functioning chlortetracycline-degrading pure bacteria strain was isolated from the sludge of the chlortetracycline pharmaceutical factory. The strain was named ZL-1 and identified to belong to gram-negative bacteria and Acinetobacter sp. based on the morphological observation, gram staining and 16S rDNA sequencing identification. Furthermore, the effects of carbon source, temperature, pH, initial chlortetracycline concentration and inoculation amount on the chlortetracycline degradation performance of ZL-1 were analyzed by the orthogonal test. The results showed that the temperature, inoculation amount, and initial chlortetracycline concentration had a greater impact on the chlortetracycline degradation efficiency. Based on the results of the orthogonal test, the chlortetracycline degradation conditions of the bacteria were optimized by the response surface method, and the optimal conditions for degrading chlortetracycline were determined. The optimal conditions were the initial chlortetracycline concentration 134.864 mg/L, the temperature 34.409 ℃, and the inoculation concentration 5.223%. Under the optimal conditions, the actual degradation efficiency of chlortetracycline was 93.70%, whereas the predictive value was 93.723%, indicating that the proposed model had high accuracy.