Resumo. The demands for energy and the scarcity in fossil fuel are constantly increasing. This has resulted in the search for sustainable, renewable, and low cost biofuel that has triggered the search for potential bioenergy crops. Aquatic plants that can grow rapidly with minimum resources and can produce biomass in bulk amounts are driving the attention of scientists and researchers throughout the world. The production of biofuels from such organic materials and waste components can result in developing of sustainable alternative that will not only be beneficial to the environment but also to public health. In this study, one such aquatic macro algae Azolla pinnata proved to be potential source for biofuel production. The evaluation of its growth was done and trans-esterification of Azolla pinnata lipid was carried out to produce biofuel. The species have a unique combination of physical, chemical and nutrients composition that makes it a boon to mankind. This macro algae was subjected to series of laboratory testing and evaluation for its characterization such as acid value test, trans-esterification, fatty acid methyl esters (FAMEs) test, gas chromatography which showed the feasibility of algal based biofuel. The comparison of properties of extracted biofuel (physicochemical) from Azollla pinnta was done with standardized ASTM D6751 values. The outcome of produced biofuel was very close to conventional fuel.