This paper presents an automatic spectrofluorimetric method (flow injection analysis spectrofluorimetry) for detecting hydroxyl radicals. Based on H2O2 catalyzed by Co2+yielding HO•, the method utilized sodium terephthalate to trap hydroxyl radicals and obtained sodium 2-hydroxyterephthalate by aromatic hydroxylation, which resulted in an increase in the fluorescence intensity. The relative fluorescence intensity was proportional to the concentration of hydroxyl radicals. Hydroxyl radicals could be determined indirectly, based on the increase of fluorescence. It was a simple, rapid, precise, sensitive and automatic technique for the determination of HO•. The relative standard deviation of 11 determinations was 0.57%. The method can be used to sieve antioxidant medicines and will have theoretical and practical guidance on the mechanism of hydroxyl radicals-damaging biology.