Abstract: A new biofuel like biodiesel applicable to diesel engines is obtained by the enzymatic ethanolysis reaction of sunflower oil with ethanol, in free solvent media, by using BIOLIPASE-R®, a low cost powdered enzyme preparation containing obtained  lipases from a strain of Rhizopus oryzae. This is a multipurpose additive from Biocon®-Spain, used in food industry. This biofuel integrates glycerol as monoglyceride (MG) together to two fatty acid ethyl esters (FAEE) molecules by the application of the 1,3-selective lipases. Thus, this process minimizes waste generation and maximizes efficiency of the process because no residual glycerol is produced. Response surface methodology (RSM) is employed to evaluate the main reaction parameters (reaction temperature, oil/ethanol ratio and pH) on the conversion of sunflower oil. Water content and amount of lipase were also investigated. Regarding the results, we found that operates optimally with a water content of the reaction medium of 0.15 %, 0.05 - 0.1 % lipase by weight relative to the weight of oil used, 20 °C, volume ratio (ml/ml) oil/ethanol 12/3.5 and pH 12 (by addition of 75 µl of 10 N NaOH dissolution). These achieved results have proven a very good efficiency of the biocatalyst in the studied selective process.