Background: As one of the most important economic crops, tobacco products have a long history and dominate the development of the world economy. Pectin, as a complex colloidal substance widely present in plant cell walls, its content is an important factor affecting the safety of tobacco smoking. Objective: This study aimed to analyze the content and structure of pectin in tobacco samples Methods: In this study, tobacco pectin was extracted by ultrasonic-assisted ionic liquid extraction, and the 13C MultiCP/MAS NMR spectral analysis of pectin was conducted. Results: The type of extractant, duration of ultrasonication, extraction temperature, and solid-liquid ratio were optimized. Under the conditions of using 1-Butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) as the extractant, the solid-liquid ratio of 1:20 g/mL, and the ultrasonic power of 600 w for 30 min at 30°C, the yield of 23.7% of tobacco stem pectin and the purity of 54.2% could be obtained. The optimized MultiCP sequence parameters, with 10 CP cycles of 1.0 ms and the repolarization time of 50 ms could obtain high-resolution spectra within a time of 1.0 h. The C-6 peaks of the pectin in spectra were fitted using the spectral deconvolution technique and calculated the methylesterification (DM) of the tobacco pectin, which was generally less than 50% and belonged to the low methyl esterification pectin. The pectin content of the tobacco sample was calculated using the standard curve method with the addition of dimethyl sulfone (DMS) as an internal reference. The results of this method were consistent with the colorimetric method. Conclusion: The 13C MultiCP/MAS NMR method has the advantages of being green, fast, and accurate and provides a new technical tool for quantitative and qualitative studies of cell wall substances in tobacco samples.