Adulterated yohimbine (YHB) in food poses a risk to public health, making it imperative to develop fast and sensitive detection methods. In this study, computational-chemistry-based prediction was employed to design YHB haptens for generating the high-affinity monoclonal antibody Yohi-4A7, which exhibited an optimal half-inhibitory concentration (IC50) of 1.69 ng/mL against YHB. This antibody was utilized in constructing a gold nanoparticle-labeled immunochromatographic assay (ICA) to influence significant analytical parameters. The ICA presented cut off values of 5.0 ng/mL for aphrodisiac liquor and 5.0 ng/g for capsules, with detection limits of 0.56 ng/mL and 0.88 ng/g, quantification limits of 1.96 ng/mL and 2.58 ng/g, and satisfactory accuracy reflected by recovery rates ranging from 96.0 to 120.7%, respectively. Analysis of 26 blind samples using the ICA, followed by comparison with liquid chromatography-tandem mass spectrometry results, revealed strong concordance between the two techniques. This work provides a reliable and efficient method for YHB detection in dietary supplements, fulfilling a critical requirement for ensuring food safety and protecting public health.