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Fluorescence and UV-Vis spectra of ofloxacin (OFL) in sulfuric acid were studied. In the present paper, a new protonation state of OFL was observed. In hydrochloric acid, OFL produced bright green fluorescence upon excitation by UV radiation. The maximal emission wavelength of OFL is about 505 nm. However, OFL produces violet fluorescence when dissolved in concentrated sulfuric acid. The maximal emission wavelength changes into 400 nm. Further analysis demonstrated that the above changes arise from the variation of protonation states of OFL molecule. In dilute sulfuric acid, OFL accepted one proton, resulting in a protonation state that is similar to the OFL molecule dissolved HCl solution. The corresponding fluorescence band occurs at 505 nm. In concentrated sulfuric acid solution, OFL might accept additional protons. As a result, the size of the conjugated system is reduced and the fluorescence band exhibits a blue shift. In sulfuric acid of moderate concentrations, two bands at 505 and 400 nm respectively were found in the fluorescence emission spectra, indicating that OFL in two different protonation states coexists in the solution. In addition, both excitation band in excitation spectra and absorption bands in UV-Vis spectra exhibit red-shifted with the decrease in the concentration of sulfuric acid. Based on the above result, OFL can be used as a spectral probe to reflect the variation of H+ in strong acid environment.