Abstract: Stable isotope labeled internal standards combined with isotope dilution mass spectrometry can offer an analytical method with good accuracy and precision for the detection of sodium cyclamate. A novel deuterium-labeled sodium cyclamate was synthesized from cyclohexanone using heavy water (D₂O) as deuterium source. The synthesis procedure featured key steps such as hydrogen-deuterium exchange, reductive amination, and sulfonylation reaction, and had the advantages of cheap and easily available starting materials, less side reactions and high isotope abundance. The deuterated product was confirmed by nuclear magnetic resonance (¹H NMR) and tandem mass spectrometry (MS) characterization with more than 99 atom% deuterium isotope abundance, which can be used as stable isotope-labeled MS internal standards for qualitative and quantitative analysis.