Ensifentrine was approved for marketing by the U.S. Food and Drug Administration (FDA) in June 2024. To date, however, the literature has provided only the raw NMR spectral data of this drug without detailed signal assignments, which hinders impurity identification and quality control for ensifentrine. Furthermore, the compound contains multiple quaternary carbon and nitrogen atoms, presenting challenges in assigning NMR signals. In this study, we employed a Bruker Avance III 400 MHz NMR spectrometer to acquire ¹H, ¹³C, and two-dimensional NMR spectra of ensifentrine, and fully assigned all ¹H and ¹³C signals. Notably, unlike previous reports, we utilized DMSO-d₆ as the solvent, which enhanced sample solubility, permitted unambiguous identification of the coupling relationships of active hydrogen signals, and yielded well-resolved two-dimensional NMR spectra. This work provides a reference for NMR-based structural analysis, content determination, and quality control of the active pharmaceutical ingredient (API) ensifentrine.