Chlorimuron-ethyl is a sulfonylurea herbicide with broad-spectrum weed control characteristics, low utilization rate, relatively high persistence in the soil. Chlorimuron-ethyl has been widely used world-over, and strategies for its removal have attracted increasing attention. Microbial degradation is considered the most acceptable dissipation method. We obtained the best biodegradation conditions using response surface methodology. Through the cleavage of the sulfonylurea bridge, we proposed a metabolic route for chlorimuron-ethyl degradation. Under these conditions (pH 6, 30 °C), Irpex lacteus could degrade 72.40% of the initially supplemented 40 mg L−1 chlorimuron-ethyl within 7 days. The half-life of chlorimuron-ethyl after inoculation was fairly short (4.696 days). The biodegradation rate of chlorimuron-ethyl by Irpex lacteus was 56.1%, which was higher than that of Phlebia sp. (50.8%) and Funalia trogii (25.4%). The pH value has an impact on the free state of the substrate molecule and the dissociation state of the enzyme molecule. The biodegradation rate was the highest (58.5%) at a pH value of 6. When the temperature was 30 °C, 56.3% of chlorimuron-ethyl was eliminated. As the temperature increased, the biodegradation rate of chlorimuron-ethyl by white-rot fungi decreased. Based on the results of LC–MS analysis, a metabolic route for chlorimuron-ethyl biodegradation was proposed. The fragment at m/z 161 (2-amino-4-chloro-6-methoxypyrimidine) originates from the cleavage of the C-N bond of the sulfonylurea bridge, while generating ethyl 2-sulfamoyl benzoate. The fragment at m/z 202 (2-sulfamoyl benzoic acid) corresponds to the ethyl group lost from ethyl 2-sulfamoyl benzoate. Due to the different secreted enzymes, there was a gap between the three strains in the degradation efficiency of chlorimuron-ethyl. The degradation rate of the herbicide by Phlebia sp. was the highest (61.7%), while by Irpex lacteus was the lowest (42.7%). Three white-rot fungi could degrade chlorimuron-ethyl in malt extract. LC–MS analysis indicated that the cleavage of sulfonylurea bridge through Irpex lacteus mediated the degradation of chlorimuron-ethyl. And, inoculation with white-rot fungi enhanced chlorimuron-ethyl degradation in aseptic soil samples. This is the principal report revealing that white-rot fungi can evacuate sulfonylurea herbicides, demonstrating that white-rot fungi will give novel ideas into the biodegradation of herbicides.