pH is a fundamental physiological parameter related to myriad applications, therefore great efforts are continuously made to develop high performance pH indicators. Here, we exhaustively investigated the pH response of an acridine analog, 9-acridinecarboxylic acid with steady state absorption and fluorescence spectroscopies, picosecond resolved fluorescence spectroscopy, and femtosecond resolved transient absorption spectroscopy. The fluorescence intensity ratio (R_450/525) of 9-acridinecarboxylic acid showed a 7-fold change in the physiological pH range (pH ∼ 5–8), and it can be served as an excellent ratiometric and lifetime pH indicator. Analysis of the picosecond and femtosecond-resolved spectra combined with theoretical calculation revealed that 9-acridinecarboxylic acid existed in zwitterionic form at acidic condition with emission centered at 480 nm and lifetime of about 2.1 ns and anionic form at alkaline condition with emission centered at 430 nm and lifetime of about 6.6 ns. As pH decreased, the protonation of heterocyclic N atom resulted in the redshift of fluorescence peak and shortening of the fluorescence lifetime. Moreover, 9-acridinecarboxylic acid exhibited good selectivity, photostability and excellent biocompatibility and was successfully applied for intracellular pH imaging by ratiometric fluorescence.