The zinc ion has a key role in a variety of physiological and pathological processes. As a consequence, the development of sensitive and reliable methods to monitor the presence of zinc ions in cells and organisms is of great importance to biological research and biomedical applications. This protocol describes detailed procedures for the five-stage synthesis of a zinc ion-selective, cyanine-based fluorescent probe, CTMPA, from 2,6- bis(hydroxymethyl)pyridine. In addition, we describe its applications in the detection of Zn 2+ released during apoptosis in cells and endogenous Zn 2+ in living zebrafish. Notably, the use of CTMPA enabled our research group to monitor for the first time the presence of zinc ions in neuromasts of zebrafish via fluorescence. The approximate time frame for the synthesis of CTMPA is 4-5 d, and for its use in bioimaging is 8-10 h for cells and 2 h for zebrafish.