Abstract: In this study, butaselen-2, 6-dimethyl-β-cyclodextrin inclusion complexes were prepared by saturated aqueous solution method to improve the solubility of butaselen, so as to obtain its injection solutions. The content of butaselen in the inclusions was determined by high performance liquid chromatography (HPLC), and then the preparation process was optimized by orthogonal design using the inclusion ratio as an indicator. X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) were used to verify the structure of the inclusions. The effects of the inclusions on the solubility and stability of butaselen were also investigated. The results showed that the optimized preparation process with a mass ratio of 1:340, an encapsulation time of 3 h and an encapsulation temperature of 70 ℃ resulted in an encapsulation ratio of (91.24 ± 0.42) %, and the results of XRD, FTIR and SEM demonstrated the formation of inclusion complexes. The developed HPLC method is rapid, simple, accurate, applicable, specific and reproducible for the determination of butaselen content in butaselen cyclodextrin inclusion complexes, which can lay the foundation for the development of new butaselen dosage forms and clinical applications and provide technical support.