This work aimed to maximize soybean oil hydrolysis mediated by Penicillium citrinum whole cells with high lipolytic activity. The effects of pH and agitation were evaluated in the reaction, and parameters such as temperature and oil mass ratio of sodium phosphate buffer were optimized through the response surface methodology (RSM). Whole cells were obtained by submerged culture in medium containing olive oil as the inducing agent and were used as a biocatalyst in the reaction medium in a proportion of 10% (w/w). The experimental results were effective and showed that , the degree of hydrolysis was maximized at a pH 8.0, 170 rpm of orbital agitation, temperature of 37°C and 20% of oil buffer mass ratio.