Norfloxacin (NOR) is a synthetic antibiotic and antitumoral drug of the class of fluoroquinolones. The electric charge of this molecular ion is mainly determined by the protonation equilibrium of two functional groups: the carboxyl of the quinolone heterocycle, and the distal amine of the piperazinyl group. The equilibrium of species with different charge distributions influences both the spectroscopic properties and pharmacological activity of the drug: NOR is positively charged in acidic medium, neutral or zwitterionic in neutral medium, and negatively charged in basic medium. In the present work, we investigated the spectroscopic properties of norfloxacin using UV-vis optical absorption, and steady state and time-resolved fluorescence in different aqueous solutions and organic solvents. Photophysical parameters such as molar absorption coefficients, Stokes shifts, quantum yields, and fluorescence lifetimes were obtained. The Stokes shift and the emission frequency were analyzed in 16 solvents, mainly grouped as protic and aprotic solvents, taking into account general effects of the solvents, such as orientation polarizability, and specific effects. It was observed that, although the UV-vis absorption spectra do not present substantial difference in most solvents, the Stokes shift and specially the fluorescence quantum yield are strongly affected by the solvent. Lippert plots of the Stokes shift and the emission frequency versus solvent orientation polarizability were constructed. Even introducing a correction for photoinduced charge transfer, according to the Weller’s theory, a good correlation was not found. On the other hand, a good correlation was found between the Stokes shift and the parameter ET(30), of the empirical scale developed by Reichardt and based on the strong solvatochromism of the betain 30 dye. Fluorescence decays in different solvents were obtained using time correlated single photon counting (TCSPC) and were fitted with the expression for lifetime distribution around a single lifetime, or using a multiexponential expression in the case of more than one lifetime. Optical absorption and fluorescence of NOR were also studied in binary mixtures of solvents: ethanol-buffer and DMSO-buffer, with pH 4.2 and 7.5 buffers. The curves of the fluorescence intensity as a function of the proportion of buffer in the mixture are characteristic of specific solvent effects, and point out that the protonation-deprotonation equilibrium of the amine and carboxylic groups plays a fundamental role in determining the quantum yield and the Stokes shift. The results showed that the fluorescence of NOR is an important spectroscopic sensor to explore interactions with biologically relevant molecules, since it is particularly sensitive to small amounts of organic solvents, especially at physiological pH.