Biodiesel manufacture is one of the advanced and technical areas for researchers as it is conquering the world because of petroleum shortage and increasing rate together with the environment friendly nature. Biodiesel define as the mono-alkyl esters of fatty acids prepared through trans-esterification reaction. The use of biodiesel is increasing rapidly worldwide due to its positive impact on diesel engines, combustion processes, and pollution formation. Biodiesel is a wellknown derivative of long-chain fatty acids that can be obtained from renewable sources such as vegetable oils or animal fats. It can be effectively used in diesel engines. The process of preparing biodiesel from raw materials, such as cottonseed oil, involves the trans-esterification of fat, followed by biodiesel characterization testing. Cottonseed oil, non-edible oil, was chosen as the raw material for this experiment, as it does not contribute to food versus fuel conflict. Methanol was used as a catalyst in the trans-esterification process for biodiesel preparation. However, the optimum conditions for biodiesel production are suggested in this paper, a maximum of 77% biodiesel was produced with 20% methanol in presence of 0.5% sodium hydroxide. Furthermore, after the preparation of biodiesel, for its identification, numbers of test was done and the prepared biodiesel was verified. Keywords:- Biodiesel, Cotton-Seed, Transesterification, Physicochemical Properties of Biodiesel.