Ethanol producing bacteria are useful in industrial production of biofuel. There are interesting for screening of active bacteria from natural resources and introduce to biofuel industries. The present study aims to isolation of ethanol producing bacteria with characterization, optimization, and evaluation of their ethanol productivity. Samples from various fruits, plant saps and soils were screened for isolation of ethanol producing bacteria then evaluate to find the highest ethanol producer. Of all the 37 ethanol producing isolates, 6 highest producers were selected for characterization. Bacterial growth and ethanol production conditions were optimized based on pH, temperature, agitation, time and initial glucose concentration. Most isolates were occurred single or in pairs. All of isolates were motile and catalase positive but failed to hydrolyze gelatin and produce H2S. Among them, Zym6 was exhibited highest ethanol yield 6.28 gL-1 with optimum pH 6 and growth temperature 35 ˚C. In addition, Zym5 and Zym6 were exhibited highest ethanol yield 19.52 gL-1 and 18.75 gL-1 with xylose and tryptophan, respectively. Thus the optimum condition for ethanol production was a medium composed of pH 6, growth temperature 35 ˚C for 24-48 hours and xylose and tryptophan as carbon and nitrogen sources.