Reaction conditions were optimized and the donor substrate selected for maximizing the reaction yield and productivity of the enzymatic synthesis of hexyl-β-galactoside with β-galactosidase from Aspergillus oryzae. We independently studied the effect of water content, type of cosolvent, temperature, donor substrate concentration, and leaving group of the donor substrate on the yield and productivity of hexyl-β-galactoside synthesis. Reaction yield was maximum in the medium with 70% water content and acetone as cosolvent, corresponding to a water activity of 0.94. Temperature and donor substrate concentration had very little effect on the yield. The leaving group of the donor substrate was the most relevant variable. Lactose, lactulose, o-nitrophenyl-β-D-galactopyranoside, and propyl- and butyl-β-D-galactoside (the last two are enzymatically synthesized from lactose) were evaluated as donor substrates. Use of propyl- and butyl-β-galactoside as donor substrates allowed us to increase the product yield by 683% and 716% [vs. lactose (0.06 mol/mol)]. Because propyl- and butyl-β-galactosides can be synthesized at low cost from lactose, using them in a two-step process could be much better alternative than a one-step process with lactose; leading to a theoretical global molar yield of 0.41 and 0.37 mol/mol from propyl- and butyl-β-galactosides, respectively.