L-Ascorbyl palmitate (L-AP) is recognized as a lipid-soluble antioxidant and has been approved for use in infant food. In response to the problems of high pollution in the chemical synthesis method of L-AP and the limited efficiency of enzymatic synthesis, a new method of catalyzing the esterification synthesis of L-AP using immobilized lipase ANL-MARE was explored. L-ascorbic acid (L-AA) and palmitic acid (PA) were used as substrates, and tert-butanol was used as the reaction solvent. A highly efficient process for the preparation of L-AP using ANL-MARE catalysis was established. Through single-factor and orthogonal experiments, the optimal synthesis process of L-AP was determined, under which a molar ratio of L-AA to PA of 1:7, an immobilized lipase ANL-MARE mass concentration of 50 g·L-1, a 3Å molecular sieve mass concentration of 250 g/L, a reaction temperature of 45 ℃, and a reaction time of 7 h were employed. Under these conditions, a conversion rate of L-AP of 90.47% was achieved, and a relative purity of 70.64% was obtained. Moreover, after continuous catalysis for 10 batches under the optimal process, a conversion rate of the immobilized enzyme ANL-MARE was maintained above 70%, and significantly better operational stability and reusability were demonstrated compared to the commercial enzyme Novozym 435. L-AP was separated and purified by column chromatography, and a purity of 92.24% was achieved. In summary, the advantage of efficient catalytic synthesis of L-AP is exhibited by the immobilized enzyme ANL-MARE. A new theoretical basis and process foundation for the large-scale enzymatic production and industrial application of L-AP are provided by this research.