Biodiesel drawn attention as a non-toxic, biodegradable and renewable soure of fuel and energy with significantly lower exhaust emissions of particulate matter and green-house. Lipases (EC 3.1.1.3) are widely employed to catalyze hydrolysis, alcoholysis, esterification and transesterification of carboxylic esters. Lipases have excellent catalytic activity and stability in non-aqueous media, which facilitate the esterification and transesterification process during biodiesel production. The aim of this work was to develop a methodology of production of lipase, in order to find a solution that promotes the production of the enzyme in a competitive price and so providing similar conversions to the alkaline catalysis. Initially was realized a fractional design 25-1 to evaluate the influence of each selected reagent for the medium of the production of the enzyme. After defining this step was realized the first tests of optimization of the conditions of culture, as well as the tests of pre-purification of the enzymatic extract always seeking the application to the production of biodiesel. Enzymatic activity was measured from the hydrolysis of olive oil emulsified. To determine the lipolytic stability of the extract in methanol and ethanol, in different concentrations of solvents (25 at 85% v/v), determining the residual activity according to standard procedure. To evaluate the capability of transesterification of the enzymatic extract was tested. The lipase obtained from the strain Candida rugosa NRRL Y-95 / NRRL Y-1496, presented a strong potential to application in industrial production of biodiesel, due to the high stability of the utilized solvent and activity of transesterification.