Terpyridine (tpy) ligands are popular building blocks to bind metal ions. Several tpy ligands with different substituents were synthesized and examined for their binding with zinc cation. The study revealed a large substituent effect on the zinc binding-induced fluorescence quenching. With the aid of a liquid nitrogen Dewar, the tpy molecules were frozen to their ground-state conformation, preventing (or minimizing) molecular reorganization in the photoinduced excited state. This allowed us to detect the fluorescence spectra from the locally excited state (having a minimum of charge transfer interaction) and the temperature-dependent fluorescence. The fluorescence response to low temperature provided useful information about the intramolecular charge transfer (ICT) interaction between the donor and acceptor groups. Furthermore, a strong donor substituent (such as Me2N) played an essential role in observed fluorescence quenching. The study also provides a useful example to elucidate the ICT mechanism by using low-temperature fluorescence spectroscopy.