Environmental friendly (bio-) chemical synthesis reactions are getting ever more important for the production of ecofriendly products. In this study a reaction system based on a deep eutectic solvent consisting of choline chloride and various sugars, was used for the enzymatic synthesis of glycolipids. A wide variety of potential substrates were tested for the enzymatic glycolipid synthesis in DES, including various sugars and one anhydro-sugar in combination with up to 9 different fatty acids. It was proven that the described DES-based solvent system is suitable for the enzymatic synthesis of tailor-made glycolipids. The proposed lipase-catalyzed acylation of the different sugars, which are substrate and part of the solvent at the same time, was detected by TLC-MALDI-ToF, ESI-Q-ToF and additional MS/MS experiments. As an example of all the synthesized glycolipids, fragments and m/z signals of the expected glycolipid arabinose-4-O-laurate was detected, and its chemical structure was elucidated via NMR.