The FDA approved ensifentrine for marketing in June 2024. Existing literature only provides nuclear magnetic resonance NMR spectral data for this drug, lacking detailed atomic assignments. This hinders impurity identification and quality control for ensifentrine. Furthermore, the compound contains multiple quaternary carbon and nitrogen atoms, presenting challenges in assigning NMR signals. This article uses the Bruker Avance III 400 MHz NMR spectrometer, by obtaining the ¹H, ¹³C, and two-dimensional NMR spectra of ensifentrine, the ¹H and ¹³C signals of this compound were fully assigned. Unlike existing literature, this study employs DMSO-d₆ as the solvent, enhancing sample solubility to accurately identify coupling relationships of active hydrogen signals while rendering the signals in the two-dimensional NMR spectrum clearly distinguishable. This provides a spectroscopic basis for the structural analysis, content determination, and quality control of ensifentrine active pharmaceutical ingredient (API) based on NMR spectroscopy.