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.