n-3 polyunsaturated fatty acids (PUFA)-rich triacylglycerols (TAG) with many beneficial effects are still difficult to be synthesized efficiently and rapidly by current synthetic techniques. This study reports the fatty acid specificity of immobilized MAS1 lipase and its efficient synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in natural deep eutectic solvents (NADES) systems. Immobilized MAS1 lipase showed the highest preference for capric acid [C10:0, the highest specificity constant (1/α)=1] whereas it discriminated strongly against docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) due to their lowest specificity constants (1/α=0.19 and 0.2). Moreover, the highest n-3 PUFA-rich TAG content (55.8%) with similar n-3 PUFA composition to the substrate was obtained in choline chloride/glycerol (CG) system. There was a 1.38-fold increase of TAG content in CG system compared with that in the solvent-free system. Interestingly, immobilized MAS1 lipase exhibited no regiospecificity in the solvent-free and various NADES systems. Besides, the potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of glycerol with n-3 PUFA in NADES systems was described. It was found that the use of NADES as solvents could greatly enhance TAG content, and make it easy to separate the product. These results indicated that immobilized MAS1 lipase is a promising biocatalyst for the efficient synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in NADES systems.