The proteolysis property of transthyretin (TTR) was believed to be important for physiological processes and have therapeutic potential. In the present study, we interchanged the corresponding sequences of Zn 2+ binding motif between human and Crocodylus porosus TTRs. The kinetic parameters of the proteolytic activity towards amyloid beta 1-42 (Aβ 1-42 ) of the mutated TTRs were determined by using a sensitive fluorescence-based method, and compared with the wild types. The results showed that C. porosus TTR with Zn 2+ -binding motif sequence of human TTR (hu-motif/crocTTR) has apparent K m (K app m ) of 118 ± 13 µM which was significantly higher than those for human TTR (53.6 ± 1.5 µM), C. porosus TTR (crocTTR) (30.2 ± 4.0 µM), and human TTR with Zn 2+ -binding motif sequence of C. porosus TTR (croc-motif/huTTR) (53.4 ± 0.87 µM). In addition, the catalytic efficiency (V max /K app m ) of croc-motif/huTTR (1.16 ± 0.0066 RFU min −1 µM −1 ) was significantly higher than human TTR (0.639 ± 0.0085 RFU min −1 µM −1 ) but similar to those of crocTTR (1.11 ± 0.082 M −1 min −1 ) and hu-motif/crocTTR (0.894 ± 0.031 RFU min −1 µM −1 ). The obtained results provided insight of the influence of the Zn 2+ -binding motif sequence and the evolutionary change structure on the proteolytic activity towards Aβ of TTR and useful information for designing a therapeutic TTR.