Nicosulfuron, one of the most widely used selective herbicides in corn field, can effectively control annual and perennial grass weeds, sedges, and some broadleaf weeds. The residual phytotoxicity of nicosulfuron in soil and water has become increasingly prominent. Therefore, an efficient method for detection of nicosulfuron was critical to ensure the sustainable and healthy development of agriculture and the ecological environment. In this paper, five nicosulfuron haptens which contained carboxyl group or aldehyde groups were designed and synthesized, and an indirect competitive immunoassay was developed for the first time. The assay showed an IC₅₀ of 8.42 ng/mL and had negligible cross reactivities toward other sulfonylurea herbicides. In the spike and recovery studies, the recovery rate from soil samples was 95 %-104 %, and that of wheat roots was 92 %-98 %, which showed a good correlation with LC-MS analysis for nicosulfuron. The immunoassay was then used to quantify nicosulfuron concentration which could cause the obvious phytotoxic symptoms to wheat. Obvious symptoms of nicosulfuron phytotoxicity in wheat root was observed at the concentration of 0.068 ± 0.006 mg/kg (ELISA result) which was consistent with 0.072 ± 0.007 mg/kg obtained by LC-MS. The developed immunoassay method is an effective tool for environment contamination monitoring.