Alkaline phosphatase (ALP) is an important enzyme, but direct imaging of ALP activity with high spatiotemporal resolution remains challenging. In this work, we rationally designed an iodinated hydrogelator precursor Nap-Phe-Phe(I)-Tyr(H2PO3)-OH (1P) which self-assembles into nanofibers to form hydrogel under the catalysis of ALP. With this property of concentrating iodine atoms at the locations of ALP, 1P was successfully applied for direct nanocomputed tomography (nano-CT) imaging of ALP activity in bacteria for the first time. We envision that, on the basis of this pioneering work, new hydrogelators containing more iodine atoms (e.g., five iodine atoms in 1P) will be designed for better nano-CT imaging of ALP activity with higher CT contrast in the near future.