In this paper, HPLC method was used to determine the hydroxyl radicals generated in Fe2+/O3 system. The experiment was focused on several factors about hydroxyl radical production including pH value, concentration of the trap agent, catalyst, reaction time, etc. The result showed that the best pH value and the [Fe2+]/[O3] were 4.0 and 0.1, respectively. The hydroxyl radicals generated in Fe2+/O3 system is stable with reaction time.