Herein, we report on the synthesis of a new class of novel non-ionic amphiphiles using triglycerol as a core, which is further functionalized with hydrophilic units poly(ethylene glycol) monomethyl ether (M-n: 350 and 550) and a pair of hydrophobic alkyl chains (C-18 or C-15) via chemo-enzymatic approach. Fluorescence measurements and dynamic light scattering studies showed that all of the synthesized amphiphilic systems spontaneously self- assemble in aqueous solution, which is further confirmed by the transmission electron microscopy. Encapsulation of hydrophobic moieties like Nile red and nimodipine was studied using ultraviolet-visible (UV-vis) and fluorescence spectrometer techniques. A cytotoxicity study of the amphiphiles using A549, HeLa, and MCF7 cell, which showed that all of the synthesized nanocarriers are well tolerated at the concentrations studied. The release profile of encapsulated Nile red in synthesized amphiphilic system was studied in the presence of the immobilized enzyme (Novozym 435).