Tobacco seeds, which holds oil in it was one of the major by-products of tobacco leave production in India. India was second largest producer of tobacco. Cigars, Hookah, Snuffs and Chewing tobacco are the major products of tobacco plant. Its seeds are found to be prominent source of non-edible oil. Extraction process have proved that seeds hold almost 41% of oil in its weight basis. In this work the physical as well as chemical properties of the tobacco seed oil and its methyl ester were investigated. Results were compared against other vegetable oil and their respective methyl esters. This work concludes that tobacco seed oil methyl ester might be an appropriate alternate fuel for diesel engines. Extraction of oil and its trans-esterification process were analysed in this study. The yield of oil in native and grounded seeds at different temperature was also studied in this work.