In order to provide excellent strains for microbial remediation of gasoline-contaminated soil, the gasoline-degrading bacteria were isolated and identified from petroleum-contaminated soil using LB medium as the substrate, and the gasoline degradation rate was determined by using the oil-reducing medium as the substrate. Single factor test was carried out on five strains with excellent gasoline degradation performance at culture temperature, medium pH and culture time. Based on the single factor test results, three factors and three levels of orthogonal test were carried out to degrade with gasoline. Response surface analysis of test results with gasoline degradation rate response, screening of strains with excellent degradation performance and determining the optimal conditions for the degradation of gasoline by the strain. The results showed that nine strains with gasoline degradation properties were isolated from petroleum contaminated soil. Among which Pseudomonas aeruginosa, Pseudomonas, Pleurobacter, Bordetella and Goldenella had excellent degradation performance. The optimal conditions for determining the degradation of gasoline by these five kinds of bacteria as follows:The optimum culture temperature for Pseudomonas aeruginosa, Pseudomonas, Pleurobacter, Bordetella and Gordonia is 32℃,the pH of the medium is 7.0 and the culture time is 20 h. The degradation rates of gasoline in the oil-reducing medium are 70.12%, 76.42%, 75.66%, 77.50% and 73.22%, respectively.