In the acetophenone degradation process by electro-Fenton, the variation trend of fluorescence characteristics of dissolved organic matter (DOM) in acetophenone synthetic wastewater was detected by excitation-emission matrix (EEM)fluorescence spectra. The fluorescence spectra characteristics of acetophenone were studied, and the fitting line of fluorescence intensity and acetophenone removal efficiency was discussed in detail. The results show that the locations of the two fluorescence centers of acetophenone synthetic wastewater are at λEx/Em=270/305 nm (Peak A) and λEx/Em=215/305 nm (Peak B), respectively, and the ratio of Peak A/Peak B is 1.22. In the electro-Fenton treatment process, firstly, acetophenone was decomposed into unsaturated fatty acid which had stronger fluorescence intensity, further, it was degraded into short-chain small molecular compounds which have weaker fluorescence intensity. Therefore, the fluorescence intensity of synthetic wastewater was increased at first and decreased afterwards in the entire 180 min electro-Fenton treatment process. The two-dimensional fluorescence peak at 285～375 nm of emission wavelength (at λEx=250 nm) had a good linear relationship with the removal efficiency of acetophenone, therefore, it could reflect effectively the removal efficiency of acetophenone in the whole electro-Fenton treatment process.