The pharmacokinetics of enrofloxacin in Chinemys reevesii was studied following intramascular injection and oral administration by HPLC-MS/MS. Enrofloxacin is the third generation of quinolones and the first animal-specific drug. Now, it is also the commonly used drug for reptiles. C. reevesii is a common native turtles in China, it is not only used as medicine or food, but also for ornament. It is another economic turtle species in addition to Trionyx sinensis. As the scale of farming has expanded each year, disease problem have gradually highlighted, but the technical problems encountered in clinical medicine has become the bottleneck of industrial development. At present, enrofloxacin is often used in the treatment of diseases of C. reevesii, such as respiratory diseases and urinary system diseases. However, there are few reports about pharmacokinetic of enrofloxacin in C. reevesii, and this experiment filled the gap.In this experiment, 20 healthy Chinese pond turtles were selected from a farm in Wuxi, Jiangsu Province. All turtles were female. After holding for two weeks, they were selected and followed by a single dose of enrofloxacin (10 mg/kg) by intramascular injection and forced oral administration (gastric intubation) respectively. Blood samples were collected continuously for the same subject 13 times from the jugular sinus. Samples were obtained and assayed by HPLC-MS/MS, and the corresponding pharmacokinetic parameters were calculated with non-compartmental models by Kinetic 4.4. The main pharmacokinetic parameters of forced oral administration were C_max: 7.49 μg/mL, T_max: 12 h, T_1/2λz: 99.85 h and AUC_0-∞: 531.67 μg/(mL·h); The main pharmacokinetic parameters of intramuscular injection were C_max:5.85 μg/mL, T_max: 4 h, T_1/2λz: 30.42 h and AUC_0-∞: 193.6 μg/(mL·h). The results showed that enrofloxacin was absorbed faster by intramuscular injection, but was absorbed better and comprehensivly by forced oral administration in Chinemys reevesii. And the retention time of forced oral administration was longer. So in the control of Chinese turtle disease, enrofloxacin is more suitable for oral administration. When we need to quickly achieve the effect, intramuscular injection of enrofloxacin will be better. When we need to continue to administer and the turtle metabolic state is normal, it is recommended to use forced oral administration of enrofloxacin by gastric intubation.