Biofuels have become a subject of significant interest due to rising fossil fuel prices and the need for fuels with reduced environmental impact obtained from sustainable sources, thus reducing greenhouse gas emissions. One such fuel is biodiesel obtained by enzymatically catalyzed transesterification of fatty acids derived from oils or fats in the presence of simple alcohols such as methanol. Improving the biodiesel production process also includes the use of various solvents, of which Natural Deep Eutectic Solvents (NADES) stand out. Due to its physicochemical properties, the application of NADES in the transesterification reaction provides favorable conditions for the action of the enzyme lipases with the simultaneous removal of reaction by-products, which results in obtaining biodiesel of higher purity with higher productivity. As part of this work, NADES based on betaine and urea were prepared in a ratio of 1: 3 with different water contents (30, 35 and 40 % (w/w)). The enzyme lipase, derived from Thermomyces lanuginosus, was successfully extracted in prepared NADES and as such was used in batch synthesis and biodiesel synthesis in a microreactor. The volume activity and stability of the lipase enzyme proved to be best in NADES betaine: urea 1: 3 with 40% (w / w) water. Therefore, this solvent was used in further work to investigate the influence of different ratios of methanol: oil: enzyme on the yield of the production process of fatty acid methyl esters (FAME). In an experiment conducted with a molecular oil:methanol ratio of 1:90, 27 % FAME was achieved in a batch reactor after 50 h and 7,61 % FAME in a microreactor during a retention time of 120 min.