Honey adulteration has become a prominent issue in the honey market. Herein, we used the fluorescence spectroscopy combined with chemometrics to explore a simple, fast, and non-destructive method to detect wolfberry honey adulteration. The main parameters such as the maximum fluorescence intensity, peak positions, and fluorescence lifetime were analyzed and depicted with a principal component analysis (PCA). We demonstrated that the peak position of the wolfberry honey was relatively fixed at 342 nm compared with those of the multifloral honey. The fluorescence intensity decreased and the peak position redshifted with an increase in the syrup concentration (10–100%). The three-dimensional (3D) spectra and fluorescence lifetime fitting plots could obviously distinguish the honey from syrups. It was difficult to distinguish the wolfberry honey from another monofloral honey, acacia honey, using fluorescence spectra, but it could easily be distinguished when the fluorescence data were combined with a PCA. In all, fluorescence spectroscopy coupled with a PCA could easily distinguish wolfberry honey adulteration with syrups or other monofloral honeys. The method was simple, fast, and non-destructive, with a significant potential for the detection of honey adulteration.