The chemical reaction related to the conversion of oils and lipids from vegetal and animal sources, in the presence of short chain alcohols in biodiesel (mono-alkyl esters of fatty acids) is known as the transesterification reaction, which is of great industrial interest. The enzymes are capable of catalyzing transesterification reactions of triglycerides found in vegetable oils, acting on the ester links of those compounds. The lipases of microbial origin enhance this process as industrial biocatalyst. In this work the influence of the variation of ethanol concentration in the reaction medium with soybean oil and the immobilized enzyme Lipozyme RM 1 M was studied. The experiments were conducted 2.0 g of soybean oil, 0.15 g of immobilized enzyme Lipozyme®TL 1 M, at 40 ºC under constant 20 rpm stirring, with ethanol volumes varying between 0.1 and 1.0mL. The samples were collected at 0, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours and analyzed by gas chromatography. It was observed that the synthesis of biodiesel was influenced by the concentration of ethanol, where the negative effect was attributed to the high polarity of alcohol, which causes the partition (migration) of alcohol to its solid phase (enzyme/support), resulting in the saturation of the enzyme microenvironment. The use of low concentrations of alcohol due to not having the stoichiometric amount necessary for the conversion of all the oil presented lower yields. For the reaction conditions used this work, the best results were found using 0.35 mL of ethanol in the reactive medium, reaching practically 100 % of yield in 8 h of reaction. Thus, this volume of ethanol shows to be best concentration found for the catalytic reaction of microbial lipase in the production of biodiesel from soybean oil, guaranteeing greater efficiency of the process.