The invention provides a podophyllotoxin derivate with biological activity and a preparation method thereof. The podophyllotoxin derivate is 1-phenyl-8-(3, 4, 5-trihydroxy)-8, 8a, 11, 11a-tetralin-1H-[1, 3] dioxo [4', 5': 5, 6] benzo [1, 2, 3-de] furan [3, 4-h] cinnoline-9(8aH)-ketone. The podophyllotoxin derivate is high in water solubility, safe to normal cells and obvious in inhibiting effect on tumor cells; the preparation method of the podophyllotoxin derivate is simple, safe, and easy to operate and control.