Forchlorfenuron (CPPU) is a widely used plant growth regulator in agriculture, and CPPU residue in food can cause harm to human health. Thus, it is necessary to develop a rapid and sensitive detection method for CPPU monitoring. In this study, a new monoclonal antibody (mAb) against CPPU with high affinity was prepared by a hybridoma technique, and a magnetic bead (MB)-based analytical method was established for the determination of CPPU by a one-step procedure. Under optimized conditions, the detection limit of the MB-based immunoassay was as low as 0.0004 ng/mL, which was five times more sensitive than the traditional indirect competitive ELISA (icELISA). In addition, the detection procedure took less than 35 min, a significant improvement over the 135 min required for icELISA. The selectivity test of the MB-based assay also showed negligible cross-reactivity with five analogues. Furthermore, the accuracy of the developed assay was assessed by the analysis of spiked samples, and the results agreed well with those obtained by HPLC. The excellent analytical performance of the proposed assay suggests its great potential for routine screening of CPPU, and it provides a basis for promoting the application of more immunosensors in the quantitative detection of low concentrations of small organic molecules in food.