A spin-labeled fluorescence probe (RNO˙) was employed to evaluate the antioxidant capacity of polyphenols. The hydrogen-transfer mechanism was proposed by kinetic studies using fluorescence spectroscopy, and the product was confirmed to be RNOH by HPLC-MS with little side products. The HPLC-UV/vis method devoted to the simultaneous monitoring of the RNO˙ and RNOH at 556 nm was developed, which can distinguish the antioxidant capacity of polyphenols with similar structure. The method has the potential to be used to screen antioxidants when modifying or optimizing natural antioxidants.