Sugar fatty acid nonionic surfactants have attracted great attention due to their good biodegradability, biocompatibility, and bio-based characteristics. Regioselective sugar acylation is difficult to control for chemical reactions. The immiscible nature between hydrophilic sugars and hydrophobic fatty acids also negatively affects the reaction efficiency. In the present study, a group of sugar fatty acid nonionic surfactants, ethyl di-rhamnolipids, was synthesized. Di-rhamnolipids were isolated from a Pseudomonas aeruginosa fermentation broth containing a rhamnolipid mixture with about 64 % di-rhamnolipids, and then purified by silica gel chromatography. Di-rhamnolipids were successfully ethylated at 0°C for 24 h, confirmed by HPLC-MS and 1 H-NMR analyses. Preliminary emulsification test indicated that ethyl di-rhamnolipids were a potential class of useful sugar fatty acid nonionic surfactants. The synthesis process was mild, did not require regioselective sugar acylation, and offered a new way of developing novel sugar fatty acid nonionic surfactants.