A kind of porous alumina microfibers was prepared via hydrothermal reaction, and then used to modify the carbon paste electrode (CPE). Compared with the unmodified CPE, the alumina microfibers modified CPE is more active for the oxidation of paeonol, and greatly increases the oxidation signals of paeonol. The oxidation mechanism of paeonol on the alumina microfibers modified CPE was studied. The oxidation process involves one electron and one proton. Based on the remarkable signal enhancement effects of alumina microfibers, a highly sensitive electrochemical method was developed for the determination of paeonol. The linear range is from 10 to 1000 nM, and the detection limit is 6.7 nM. It was used in the analysis of traditional Chinese medicine samples. The results obtained by this method and high-performance liquid chromatography are in good agreement.