Objective To obtain active Salmonella typhimurium strains with resistance to sarafloxacin hydrochloride in vitro and investigate the lowest concentration of sarafloxacin that can cause the resistance of Salmonella typhimurium, which could provide the basis for the evaluation of microhial resistance caused by sarafloxacin residues in food. Methods In inducing the drug resistance of Salmonella typhimurium, the concentration of standard hydrochloric acid sarafloxacin in culture media was 0 μg/ml in blank control group and 0.001, 0.002 5, 0.005, 0.025, 0.05, 0. 1 μg/ml in 6 experimental groups respectively, and also a solvent control group (NaOH) was set. The MIC of induced strains was tested by NCCLS methods and the bacterial resistance was determined according to ≥8 × MIC（0.25 μg/ml）. PCR amplification of the quinolone resistance-determining region of gyrA in resistant Salmonella typhimurium was performed and the amplicons subjected to a pyrosequencing protocol was identified for 5 common mutation sites, Ala67, Gly81, Asp82, Ser83 and Asp87. Results The MIC of the 10th generation of Salmonella typhimurium induced by 0.005 μg/ml of sarafloxacin hydrochloride was increased 32 times, and the bacteria stopped proliferation while the inhibitory concentration increased to 1 μg/ml. Several stable, bioactive resistant strains with a mutation in gyrA at Ser83 were obtained at the 25 passage. The bacteria could not grow when the concentration was ≤0.002 5 μg/ml. Conclusion The resistance of Salmonella typhimurium could be induced by sarafloxacin at a concentration of 0.005 μg/ml, and drug-resistant strains with biological activity could be obtained several passages.