Abstract: Vitamin A shows a variety of physiological activities, such as maintaining normal vision, improving skin and anti-aging activity. However, it is extremely unstable and easily decomposed under the action of light, heat and oxygen. In this study, Novozyme435 was used to catalyze the transesterification reaction of Vitamin A acetate (VAAE) and ethyl docosahexaenoate (EDHE). The target product (Vitamin A docosahexaenoate, VADHE) was identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS). On this basis, VADHE in the reaction system was purified by silica gel column chromatography (C18 filler). The result of HPLC shows that the purity of VADHE reached 90%. In order to increase the yield of VADHE, the transesterification reaction was optimized in organic solvent system and solvent-free system, respectively. In the organic solvent system, the conversion rate reached 40.61% after 6 h. In the solvent-free system, when 100 mg Novozyme was used to catalyze the reaction for 4 h, the conversion rate reached 56.39%. VADHE was firstly designed and synthesized in this study, and the purified VADHE can be further used in the physiological activity experiment.