There has been considerable interest in the development and production of biodegradable polymer to solve the problem of pollution caused by the continuous use of synthetic polymer of petroleum origin. Polyhydroxyalkanoates (PHAs) are known to be accumulated as intracellular inclusion in some bacteria. They are accumulated intracellularly as carbon and energy reserves under certain conditions like, in the presence of different carbon sources and other nutrients. For PHB production bacterial strains Enterobacter aerogenes and Bacillus thuringiensis have been selected in this study. The maximum PHB yield was recorded under the cell dry weight basis respectively with glucose as the sole carbon source at PH 7 and temperature 37oC for 48 hours. Bacterial strains have been screened for PHB production by Sudan Black B staining method. PHB has been extracted and purified by acetone-chloroform method. Purification, quantification and analysis of functional group of PHB has been carried out by using HPLC.