Abstract: Objective To optimize the formula of liposome podophyllotoxin chitosan film and accurately determine the podophyllotoxin content in the film. Methods Liposome podophyllotoxin chitosan films were prepared according to different formula derived from the results of orthogonal design by selecting chitosan, acetic acid and gelatin as the 3 factors for preparation, each factor including 3 levels with liposome podophyllotoxin as the blank factor. Comprehensive evaluation of the formulas was performed on the basis of the conglutination and solubility of the prepared film to select the optimal formula. The content of podophyllotoxin was determined by ultraviolet spectrophotometry at 292 nm. Results The conglutination and solubility were optimal of the film prepared from 2% chitosan, 1% acetic acid, 2% gelatin and 2.8% liposome podophyllotoxin chitosan, showing good linearity within podophyllotoxin concentration range of 5-25 μg/ml (r=0.999 89) with an average recovery rate of 99.7% (n=5) and RSD of 1.11%. Conclusion The film prepared according to the orthogonal design results conforms to the requirements in terms of conglutination and solubility, and the method we adopted is fast and accurate, yielding qualified film with censored podophyllotoxin content that distributes homogeneously.