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This work investigated the best reaction conditions to obtain biodiesel from soybean oil using Candida rugosa lipase (CRL) immobilized on p‐nitrobenzyl cellulose xanthate (NBXCel) support. The alcohols used were methanol and ethanol and the molar ratio used was 1:4 (n/n). The CRL immobilized on NBXCel support showed greater catalytic activity at pH 7.00 and at 45 °C. Thus, 23 experiments were carried out at this pH and at this temperature, varying the reaction time, type of alcohol, and amount of immobilized CRL to achieve a conversion equal to or greater than 96.5% as recommended by EN 14103. The highest conversions of methyl biodiesel were achieved using 4.31%mCRL/mNBXCel and with 3.89%mCRL/mNBXCel. Ethyl biodiesel was achieved at the highest conversion using 5.18% and 4.76%mCRL/mNBXCel. The results were explored with regression analysis to identify the best reaction conditions to obtain a good conversion, and the response surface graphs were plotted to visualize other reaction conditions. We also evaluated the number of reuse cycles of CRL immobilized on NBXCel support, to lower biodiesel production cost, and after 15 cycles of reuse the conversions obtained were above 50%, demonstrating the efficiency of CRL immobilized on the NBXCel support in catalyzing the synthesis of biodiesel. © 2021 Society of Chemical Industry and John Wiley & Sons, Ltd