In this study, a time-resolved fluorescence immunochromatographic assay (TRFICA) was developed for the rapid determination of rhodamine B (RB) in condiments. Labeling conditions for fluorescent probes, test strip coating conditions and reaction conditions were optimized. The limits of detection (LOD) of the TRFICA method were determined to be 1.9, 0.7, and 1.5 µg/kg for chili powder, chili oil and tomato sauce, respectively, and the linear ranges 3.4–22.4, 1.4–14.8, and 2.4–11.8 µg/kg, respectively. Recoveries of RB from spiked samples ranged from 87.0% to 106.1%, with coefficients of variation (CV) between 5.4% and 11.6%. Moreover, a strong correlation was observed between the results obtained by this method and those from liquid chromatography-tandem mass spectrometry (LC-MS/MS)(R² = 0.9413). This study presents a novel technical tool that supports efforts to prevent the illegal use of RB in condiments.