Anions are ubiquitously distributed not only in biological systems but also in environment. Accordingly, anion recognition and sensing have attracted increasing attention due to the important roles anion plays in biological, chemical and environmental fields. Among various anionic species, NO3- is a very important inorganic anions which has a great harm to the environment and human health. At present, methods for the determination of NO3- mainly contained electrochemical method, ion chromatography and ion selective electrode method. Although all methods have their own advantages, but also has the obvious deficiency. Such as, the reproducibility of electrochemical method is not good, and ion chromatography and ion selective electrode method usually require expensive apparatus and lengthy analytical time. As a contrast, fluorescence spectroscopy become a hot research topic in anion recognition and detection because of its high sensitivity and easy operation and other advantages in recent years. New bipyridinium salt L was designed and synthesized using pyrylium as a starting material. The molecular structure was determined by 1H NMR, 13C NMR, and high resolution mass spectrometry. The molecular recognition properties of L have been investigated through fluorescence titration experiments. The results indicate that L has sensitive and selective fluorescent response to NO3- among other different anions. Significant enhancements in the fluorescence intensity of L were observed when various concentrations of NO3- were added, while other competing anions have a quenching effect towards the initial fluorescence of solution. The fluorescence titration spectra shows that the bonding stoichiometry between receptor L and NO3- is 1 : 1 as a supramolecular complexes (1gK = 5 ± 0.02). The chemosensing properties of L were evaluated through quantum chemical calculations and the variable temperature 1H NMR titration. These results suggested that L has strong binding affinity towards NO3- with high selectivity, which may be ascribed to the specific hydrogen bonding between the L and active H atom of the bipyrydinium salts. The interaction between L and NO3- made the complex more planar compared with L giving rise to enhanced fluorescence and specific selectivity towards NO3-.