Objective To study the interaction of γ-cyclodextrin (γ-CD) and its two derivatives with rocuronium and vecuronium. Methods The interactions of γ-CD and its two derivatives HS-7, Sugammadex with rocuronium and vecuronium were characterized by Isothermal Titration Calorimetry (ITC), and the thermodynamic constant and the binding constant was determined. The binding constant and inclusion ratio of γ-CD with rocuronium and vecuronium was observed by ultraviolet-visible spectrophotometry; the binding ability of three cyclodextrins with rocuronium and vecuronium was analyzed by nuclear magnetic resonance spectroscopy COSY and ¹HNMR. Results The results of ITC showed that HS-7, Sugammadex combined with rocuronium and vecuronium in a 1:1 molar ratio, and γ-CDs combined with rocuronium in a 2:1 molar ratio, with the binding constant of HS-7 and rocuronium being (3.44±2.18)×10⁷ L/mol, HS-7 and vecuronium being (5.80±1.83)× 10⁶ L/mol, Sugammadex and rocuronium being (1.04±0.34)× 10⁷ L/mol, Sugammadex and vecuronium being (2.53±1.07)×10⁶ L/mol, and γ-CD and rocuronium being (2.84±3.41)×10⁴L/mol. The results of ultraviolet-visible spectrophotometry revealed that γ-CDs combined with rocuronium and vecuronium in a 2:1 molar ratio, with the binding constant of γ-CD and rocuronium being 6.93×10⁴L/mol, and of γ-CD and vecuronium being 5.17×10⁴ L/mol. The results of nuclear magnetic resonance spectroscopy suggested that the binding ability of HS-7 with rocuronium and vecuronium was stronger than that of Sugammadex and γ-CD. Conclusion The binding ability of HS-7 with rocuronium and vecuronium is stronger than that of Sugammadex and γ-CD, and the three methods used in this study (ITC, ultraviolet-visible spectrophotometry and nuclear magnetic resonance spectroscopy) can characterize the inclusion complex from different perspectives.