OBJECTIVE： To establish a method for the concentration determination of levofloxacin in rat plasma and compare the pharmacokinetic difference between Levofloxacin tablets and gastric floating sustained-release pellets in rats. METHODS： SD rats were randomly divided into Levofloxacin tablets group and Levofloxacin gastric floating sustained-release pellets group， with 6 rats in each group. They were given relevant medicine intragastrically 40 mg/kg （taking normal saline as solvent）， and the blood samples 0.3 mL were collected before medication and 0.25， 0.5， 1， 2， 4， 8， 12， 24 h after medication. The plasma concentration of levofloxacin in rats was determined by UPLC. The determination was performed on Waters Acquity UPLC BEH C18 column with mobile phase consisted of 0.1％ formic acid-acetonitrile （78 ∶ 22，V/V） at the flow rate of 0.3 mL/min. The detection wavelength was set at 294 nm， and column temperature was 40 ℃. The sample size was 2 μL. The pharmacokinetic parameters of rats were calculated by using DAS 3.0 software， and the difference between them were detected by F-test. RESULTS： The linear range of levofloxacin was 0.20-20.12 μg/mL， and limit of quantitation was 0.20 μg/mL. The limit of detection was 0.04 μg/mL. The intra-day and inter-day RSDs were less than 10％. The recoveries were all in line with the related requirements of quantitation analysis of the biological samples stated in 2015 edition of Chinese Pharmacopeia. Average drug concentration-time curves of single dose of Levofloxacin tablets group and Levofloxacin gastric floating sustained-release pellets group were all in line with two-compartment model after intragastric administration. The pharmacokinetic parameters cmax were （12.13±1.67） and （8.76±1.13） μg/mL； tmax were（0.86±0.15） and （2.48±0.45）h； t1/2β were（4.67±0.95） and （6.67±1.01）h； AUC0-t were （42.95±4.21） and （126.48±9.44） μg·h/mL； AUC0-∞ were （50.66±6.72） and （132.61±10.63） μg·h/mL， respectively. Compared with Levofloxacin tablets， cmax of Levofloxacin gastric floating sustained-release pellets were decreased significantly， and tmax， t1/2β， AUC and mean retention time were prolonged or increased significantly （P＜0.05）， and relative bioavailability was 294％. CONCLUSIONS： Established UPLC method is simple， specific， sensitive and precise， and can be used for the determination of levofloxacin concentration in rat plasma and its pharmacokinetic study. After levofloxacin is made into gastric floating sustained-release pellets， pharmacokinetic parameters are changed significantly， retention time is prolonged significantly and bioavailability is improved significantly.