Transthyretin (TTR) is a transporter for thyroid hormone and retinol binding protein that has recently been reported to have proteolytic activity against certain substrates, including amidated neuropeptide Y (NPY). However, the proteolytic activity of TTR towards NPY is not fully understood. Here, we used fluorescence‐based assays to determine the catalytic kinetics of human TTR towards human amidated NPY. The Michaelis constant (KM) and catalytic efficiency (kcat/KM) of TTR proteolysis were 15.88 ± 0.44 μm and 687 081 ± 35 692 m−1·s−1, respectively. In addition, we demonstrated an effect of the C‐terminal sequence of TTR. When the C‐terminal sequence of TTR was made more hydrophobic, the KM and kcat/KM changed to 12.87 ± 0.22 μm and 983 755 ± 18 704 m−1·s−1, respectively. Our results may be useful for the development of TTR as a therapeutic agent with low risk of the undesirable symptoms that develop from amidated NPY, and for further improvement of the kcat/KM of TTR.