In order to improve preparation quantity and separation degree, proportion of mobile phase was stud-ied as the main object, the preparation condition of endophytic fungus secondary metabolites was optimized for semi-preparation HPLC based on the analyzing results of HPLC. The ideal gradient elution program was as follows:first mobile phase at the ration of water and methanol was 90∶10 for 0 to 10 min, second mobile phase at the ration of water and methanol was 85∶15 for 15 to 50 min, and the last mobile phase ration of water and methanol was 20∶80 for 50 to 60 min.