Pretilachlor is a chloroacetamide herbicide, mainly used for the control of weeds and broadleaf weeds in rice, and widely used in China. For the detection of residues of pretilachlor in the en-vironment and food, a highly sensitive and specific monoclonal antibody against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47±2.35 μg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 μg/L was developed for the detection of pretilachlor residues in the environment and food crops. The specificity of the antibody was explained by computer simula-tions and experimental validation. No cross-reactivity of this monoclonal antibody to alachlor, acetochlor and metalaxyl, and it cross-reacted less than 3.0% to both butachlor and propisochlor. The limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 μg/L. The recoveries of all samples were 78.3%~91.3%. The ic-ELISA method was validated by high-performance liquid chromatography, thus, it can be used for residue detection of pretilachlor in the environment and grains.