2-(2'-Hydroxyphenyl)imidazo[1,2-a]pyridine (HPIP, 1) and its derivatives are synthesized, and their fluorescence properties are studied. Although all the compounds show faint dual emission (Φ ≈ 0.01), which is assigned to the normal and excited-state intramolecular proton transfer (ESIPT) fluorescence in a fluid solution, they generally display efficient ESIPT fluorescence (Φ up to 0.6) in a polymer matrix. The introduction of electron-donating and electron-withdrawing groups into the phenyl ring causes blue and red shifts of the ESIPT fluorescence emission band, respectively. On the other hand, the introduction of such groups into the imidazopyridine part results in fluorescence shifts in the opposite directions. The results of ab initio quantum chemical calculations of the intramolecular proton-transferred (IPT) state are well in line with the ESIPT fluorescence energies. The plots of the calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels against the Hammett substituent constants (σ) show good linearity with different slopes, which can rationalize the effect of the substituent and its position on the IPT state. Therefore, we have developed a series of HPIPs as new ESIPT fluorescent compounds and demonstrate that ESIPT fluorescence properties would be rationally tuned using quantum chemical methods.