In this study, nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC) were used for the qualitative identification and quantitative determination of a novel bisacodyl derivative illegally added to food, namely 5-chlorobisacodyl cyclopropanecarboxylate. First, NMR spectroscopy was employed to qualitatively confirm the reference standard of 5-chlorobisacodyl cyclopropanecarboxylate, and a quantitative HPLC method was subsequently developed. The extraction efficiencies of acetonitrile and methanol were investigated separately. Chromatographic separation was performed on a Waters Xbridge C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic elution using methanol:water = 72:28 (v/v) at a flow rate of 1.0 mL/min, and the detection wavelength was set at 230 nm. Under these conditions, the retention time of 5-chlorobisacodyl cyclopropanecarboxylate was approximately 16.39 min. Method validation results showed good linearity in the concentration range of 0.5–50.0 μg/mL, with the regression equation:y = 5105x ? 0.0199, r2 > 0.999The limit of detection (LOD) and limit of quantification (LOQ) were 10 mg/kg and 25 mg/kg, respectively. Average recoveries at three spiked levels in four different matrices (compressed candy, jelly, beverage, and preserves) ranged from 80.21% to 102.05%, with relative standard deviations (RSDs) of 1.59% to 5.98%. The content in the positive sample was determined to be 40 mg/kg. This work provides a rapid and reliable method for the qualitative and quantitative analysis of 5-chlorobisacodyl cyclopropanecarboxylate. As this illegally added compound has not been reported at home or abroad, it deserves close attention.