To enhance the esterification activity and stability of lipase in organic solvents, the bio-imprinted Aspergillus niger lipase combined with cross-linked aggregate immobilization was investigated, and was applied to the catalytic esterification for the synthesis of Vitamin E succinate in an N, N-dimethylformamide (DMF) solution. Lauric acid, serving as a succinic acid analogue, was selected as the bio-imprinting molecule. The catalytic activity of the resulting cross-linked bio-imprinted lipase aggregates was significantly enhanced, achieving an esterification yield of 87.4 ± 0.43% for Vitamin E succinate. Moreover, the aggregates maintained their catalytic activity over five consecutive reaction cycles in DMF. Fluorescence spectroscopy analysis revealed that bio-imprinting promoted the exposure of the lipase active sites, which corresponded with the observed increase in esterification activity. The mechanism of the substrate analogue-imprinted lipase was characterized. This study provides a theoretical foundation for improving the catalytic esterification performance of lipase as well as a process basis for the enzymatic synthesis of Vitamin E succinate.