Alpha-glucosidase (GAA), as an important glycoside hydrolase, is avital functional protein to maintain human blood glucose balance. For a long time, researchers have focused on exploring foods and drugs with inhibition of α-glucosidase activity in order to reduce blood glucose in people with hyperglycemia. However, few studies have focused on the possible effects of non-active intake of exogenous substances on the normal physiological functions of GAA. Given this, 9-hydroxyphenanthrene (9-OHPhe), a hydroxyl metabolite of a typical polycyclic aromatic hydrocarbon—phenanthrene, was selected in this study to explore its influence on GAA activity and potential mechanism. In order to obtain the binding information of 9-OHPhe and GAA, the PARAFAC method was applied to analyze the three-dimensional fluorescence spectra (EEM) data of 9-OHPhe and GAA with overlapping spectra, and the molecular docking method was used to analyze the microscopic information of their binding at the molecular level. 9-OHPhe inhibited the activity of GAA, and the corresponding IC50 value was (7.59±1.91) μmol·L-1. PARAFAC method can be effectively used to analyze the fluorescence data of GAA and 9-OHPhe systems with overlapping fluorescence spectra. 9-OHPhe can cause the static quenching of endogenous fluorescence of GAA, and they can form a 1∶1 complex with the binding constant of (8.91±0.68)×103 L·mol-1 at 298 K. The hydrophobic interactions existed between 9-OHPhe and GLN281, LEU305, ASN319, LEU321, TYR322, LEU323, TYR352 in GAA, and two hydrogen bonds with bond lengths of 2.71 and 3.05  formed for 9-OHPhe with GLN309 and ASN319. A low concentration of 9-OHPhe can stabilize the secondary structure of GAA, while too high a concentration of 9-OHPhe will destroy the structural stability of GAA. 9-OHPhe showed anobvious inhibitory effect on the activity of GAA, suggesting that the glucose balance disorder which might be caused through this pathway after the relevant exogenous pollutants entered the healthy human body was worthy of attention and in-depth study.