A novel derivative of O-propyl vardenafil was identified for the first time in a candy product with aphrodisiac claims using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The compound’s molecular structure and fragmentation pattern were deduced from fragment ion data, and further confirmed as vardenafil impurity 31, a derivative of O-propyl vardenafil, based on 1H NMR. Qualitative and quantitative detection methods of O-propyl vardenafil and vardenafil impurity 31 detection were developed using liquid chromatography-triple quadrupole mass spectrometry. Both analytes showed good linearity in the range of 1~50 μg/L. The limits of detection and quantification were 0.05 and 0.1 mg/kg, respectively. Recoveries at three spiked levels in solid drink, candy, oral liquid, and lipid matrix ranged from 87.2% to 113%, with relative standard deviations of 1.1%~8.0%. The method was applied to 234 commercial samples, in which O-propyl vardenafil was detected in 29 batches and vardenafil impurity 31 in 5 batches. Owing to its high sensitivity and accuracy, the method is suitable for qualitative and quantitative determination of illegally added O-propyl vardenafil and its derivative in various food matrices, providing a scientific basis for regulatory monitoring and investigation.