The invention relates to preparation and application of a gene and chemotherapeutic drug co-delivery anti-tumor nanoparticle of a target SR-BI (Scavenger Receptor-BI). The nanoparticle is a recombinant high density lipoprotein drug-loaded nanoparticle, comprises phospholipid, cholesterol, cholesteryl ester, apolipoprotein, cation lipid, a chemotherapeutic drug, a gene drug and the like, and is prepared by a film dispersion method. The invention aims at preparing the bionic tumor target drug-loaded nanoparticle, the preparation condition is mild, and the cost is low. The invention further provides the application of the gene and chemotherapeutic drug co-delivery anti-tumor nanoparticle. The gene and chemotherapeutic drug co-delivery anti-tumor nanoparticle has the advantages of spherical structure, good encapsulation efficiency, low cytotoxicity, good targeting, high safety, significant in-vitro anti-tumor effect and the like.