Abstract: Microbial compounds that exhibit pronounced surface and emulsifying activities are classified as biosurfactants. The effect of carbon and nitrogen sources and other physiological parameters affecting the biosurfactant production by Pseudomonas putida MTCC 2467 was studied. Different carbon and nitrogen sources were tested for biosurfactant production using minimal medium and it has been found that sucrose and ammonium sulphate were found to be the best carbon and nitrogen source. The effect of initial pH was studied by varying pH from 5.0-8.0 and maximum biosurfactant production, 2.8 g L-1 was achieved at an optimum pH of 8.0. The biosurfactant production was also monitored by measuring the liquid surface tension and interfacial tension and the results showed that the medium surface tension reduced from 74-35 mN m-1. The stability of biosurfactant was determined by varying pH, temperature, salinity and metal ions found that biosurfactant was stable at alkaline pH and higher temperature range suggesting that biosurfactant was highly stable, purified and checked for protein and lipid content and characterized by FTIR spectroscopy.