Rifampicin is a semi‐synthetic broad‐spectrum antibiotic obtained from rifamycin B. It is one of the most effective first‐line antituberculosis drugs and is widely used in clinical practice. In the present study, we describe a rapid and sensitive method for the determination of rifampin in aquatic products by stable isotope‐dilution high liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Samples were extracted with the acetonitrile, degreased by hexane, and then concentrated by nitrogen blowing. After separation using a C18 column with a mixture of acetonitrile and water as mobile phase, it was determined by HPLC–MS/MS using the stable isotope‐dilution calibration method. The performance of our method was validated. The limit of detection was 0.25 μg kg−1 and the limit of quantification was 0.5 μg kg−1. At the three spiked levels of 0.5, 1.0 and 5.0 μg kg−1, the average recoveries of rifampicin in different aquatic products were between 75.28 and 107.6%, and the relative standard deviation ranged from 0.81 to 13.23%. This method was successfully applied for the determination of rifampin in different kinds of aquatic products and rifampicin residue was found in aquatic products obtained from markets in Beijing, China.