A series of novel push-pull fluorophores based on 2H-imidazole scaffold bearing perfluorophenyl substituent and extended electron-donating conjugating π-system were synthesized. In particular, the Pd-catalyzed Suzuki-Miyaura coupling reactions were exploited as a main synthetic strategy to modify 4-(4-bromophenyl)-2,2-dimethyl-5-(pentafluorophenyl)-2H-imidazole with various electron-donating functionalities. The comprehensive photophysical studies were carried out for the synthesized fluorophores, including the analysis of absorbance and emission spectra and determination of absolute quantum yields. The designed compounds were found to have a strong emission in the range of 470–610 nm depending on both the nature of solvent used and the structure of electron-donating group attached to the aryl moiety. The pH effect of the aqueous-organic mixture buffer solution on the fluorescent characteristics of the synthesized compounds was established and the deprotonation-induced “turn-on” pH sensing mechanism supported by DFT calculations was proposed for the first time. Particularly, the sequence of deprotonation was confirmed, and free energies for all N-protonated and N-proton-free forms were calculated. No toxic effect on the human embryonic kidney cells (HEK-293) at concentration of ≤512 μM was observed in vitro experiments. In addition, new opportunities for the practical application of these molecular ensembles were demonstrated in case of fluorometric determination of the pH both in model solutions and real saliva probes, with 3-(4-(2,2-dimethyl-5-(pentafluorophenyl)-2H-imidazole-4-yl)phenyl)-9-phenyl-9H-carbazole being used.