Diacylglycerols have been extensively used in food, medicine, cosmetic, and pharmaceutical industries due to their outstanding properties as emulsifiers and stabilizers as well as their nutritional benefits to humans. This study investigated the optimization and kinetics of lipase-catalyzed synthesis of glycerol dioleates (GDO) through esterification of glycerol with oleic acid in a mixed solvent system of acetone/tert-butanol. Process variables, namely, solvent ratio, temperature, and enzyme loading, were optimized to acquire maximum conversion and selectivity. A high oleic acid conversion of 73% and GDO selectivity of 77% were obtained in an acetone/tert-butanol 70:30 (v/v) mixture at 45 °C and an enzyme load of 0.15 g. The bi substrates Ping-Pong Bi-Bi kinetic model was employed for initial rate determination, which was fitted to the experimental results. At an applied temperature of 45 °C, the Ping-Pong Bi-Bi model with no substrate inhibition gives the best fit with parameter values of Vmax = 1.21 M h–1, Km[G] = 1.81 M, and Km[OA] = 6.81 M for glycerol and oleic acid concentrations between 0.31 to 1.32 M and 1.88 to 2.13 M, respectively.