Phytosterol esters are functional compounds that can effectively reduce plasma cholesterol concentration, and have wide applications in the food industry. In this study, a simple and efficient enzymatic method was successfully applied to synthesize phytosterol oleic acid esters with fatty acids from high-oleic sunflower seed oil. Among the tested lipases, Candida rugosa lipase (CRL) exhibited higher catalytic activity in the esterification of phytosterols with fatty acids (oleic acid 84%) from high-oleic sunflower seed oil. Box–Behnken design and response surface methodology were used to investigate the influence of reaction factors on the conversion of phytosterols. The maximum conversion of phytosterols (96.8%) and yield of phytosterol esters (92%) could be obtained under optimal conditions: reaction temperature 50 °C, a molar ratio of phytosterols to fatty acids at 1 : 2.3, enzyme loading of 5.8%, isooctane volume of 2 mL and reaction time of 2 h. It was noteworthy that this enzymatic esterification method indeed expended a much shorter reaction time (2 h) than that observed in previous reports. In general, the enzymatic preparation of phytosterol oleic acid esters with fatty acids from high-oleic sunflower seed oil will be a simple and rapid method for producing unsaturated fatty acid esters of phytosterol with both higher oil solubility and oxidative stability, which is beneficial as functional food ingredients.