Pichia pastoris is a system to express a lot of foreign proteins, however the expression levels of transformants vary widely from one to one clone. In situ two-film method is a rapid method based on immunology to screen Pichia pastoris transformants expressing high-level protein, in which yeast colonies are lifted from solid culture onto a cellulose acetate film. Then a nitrocellulose film is used to capture the secreted proteins that pass through the cellulose acetate film. The proteins binding on the nitrocellulose film are stained by immunology methods. Pichia pastoris transformants expressing high-level hFL are abtained by using this method, and the expression level in liquid culture is about 20 mg/L. The result of ELISA shows that the staining intensity is correlated to the expression level in liquid culture. Western blot of culture supernatant shows obvious band at 25 ku, while the control doesn't show any band. The expression quantity increases as the inducing time increases.