Objective To compare the efficiency and quality in islet isolation and survival of islet grafts among three digestive enzymes for islet isolation so as to provide reference for enzyme selection in clinical islet transplantation. Methods Under the same conditions, digestive enzymes of Liberase TL (A1 group), Collagenase P (B1 group) and Collagenase Ⅴ(C1 group) were used for pancreas digestion in mice, with 35 mice in each group. Ficoll-400 density gradient method was adopted for islet purification. The morphology of islet cells was observed by DTZ staining, and IEQ (islet cell cluster with diameter>150 μm) and purification of islet cells were calculated. The survival of islet cells was determined by trypan blue staining. C57BL/6 diabetic mice were served as receptors, and were randomly divided into A2 group, B2 group and C2 group, with 7 mice in each group. Syngenetic islet transplantation under renal capsule was performed, and changes of blood glucose were monitored after operation. The graft samples were obtained 15 d after transplantation, and histological changes were observed with HE staining. Results The quantity of IEQ, purity of pancreas and survival of islet cells in A1 group were the highest, those in C1 group were the lowest, and there were significant differences among groups (P<0.05). Each receptor mouse obtained (900±30) IEQ islets, and there was no significant difference in blood glucose among groups before transplantation (P>0.05). The blood glucose was the lowest 2 d after transplantation, increased on the third day, then maintained at a relatively stable state, and on the thirteenth day, the blood glucose in A2 group was lower than that in B2 group, while the blood glucose in C2 group was higher than that in B2 group, with significant differences among groups (P<0.05). HE staining revealed that the appearance of islet in A2 group was intact, the cytoplasm and nucleus were clearly discriminated, the survival status was favorable, and the inflammatory infiltration was not significant. The appearance of islet in B2 group was round or oval, the cells were multinuclear, with understained cytoplasm, and the inflammatory cell infiltration was more significant than that in A2 group. The inflammatory cell infiltration was significant in C2 group, with damaged islet and irregular morphology. Conclusion Compared with Collagenase P and Collagenase Ⅴ, Liberase TL can improve the quantity, activity and function of islet cell clusters in pancreas digestion in mice.