Abstract: Micro-organisms was to isolated and screened from different samples which could grow at a high butanol concentration,through repeated the determination of the relative growth rate in the medium what was increased the adding amount of butanol continuously, After repeatedly, Chose strains with higher relatively growth rate and drew the resistance curve, analysis the butanol concentration of strains can tolerate. The strains were identified by physiological and biochemical experiments and 16 S rDNA series analysis, combined with the bacteria and bacterial colonies characteristics, establish the species of the strains.The concentration of butanol that two bacterial strains toleranced were all in 3% (v/v). The 16S rDNA sequences analysis and biochemical Properties test result of two strains was consistent, which revealed that A37 and A50 was Enterococcus faecalis and Lactobacillus fermentum, respectively. Results showed that two bacterial strains has high butanol tolerability, for the next step to the strains of metabolic engineering transformation production butanol laid a foundation.