The invention discloses a preparation method and an application of an active natural super-molecular photosensitizer. The active natural super-molecular photosensitizer is prepared with sterol carriernanoparticles as a carrier and a chlorine photosensitizer Ce6 as a drug. A natural product with anti-cancer activity is used as a nanocarrier to carry the photosensitizer, on one hand, the prepared nano complex photosensitizer has significantly improved water solubility, enhanced stability, increased reactive oxygen yield, improved cytophagy capacity and efficient in-vitro and in-vivo anti-canceractivity; and on the other hand, sterol products with anticancer activity themselves have an obvious synergistic anti-cancer effect after being combined with the single photosensitizer, meanwhile, sterol substances have excellent biocompatibility and biodegradability, and efficient and safe anti-tumor therapy is finally ensured.