Mono-olein is the most widely used emulsifier in food and pharmaceutical industries. It was produced by direct esterification of oleic acid with glycerol using immobilized Staphylococcus simulans lipase. The best mono-olein yield was achieved with CaCO3 as support. No organic solvent was necessary to solubilize the substrates. This allowed the use of a reaction medium solely composed of the necessary substrates. Response surface methodology based on three-level, three-variable central composite rotatable designs was employed to optimize three reaction variables: the amount of lipase, the initial added water and the oleic acid to glycerol molar ratio. The optimum conditions predicted for achieving 70.6% yield of mono-olein were as follows: an amount of lipase of 100 IU, an initial added water of 5% (w/w) and an oleic acid to glycerol molar ratio of 0.2. The experimental results confirmed the predicted values.