In order to conceal the unpleasant smell of pharmaceutical raw materials of Penicillin V potassium, we use β-cyclodextrin (β-CD) as a drug carrier. The Penicillin V potassium β-CD inclusion complex was prepared by saturated aqueous solution method; the characterization of penicillin V potassium β-CD inclusion complex is determined by scanning electron microscopy, Differential Scanning Calorimeter (DSC) and X-ray Diffraction (XRD), the formation constants of the complexes were determined by UV spectrophotometry; based on the results obtained from the orthogonal experimental design, the optimum preparation process are summarized as follow: β-CD: Penicillin V potassium = 4: 1 (molar ratio), stirring temperature 50 centigrade, stirring time 12h, the encapsulation efficiency is 29.40%.