The present invention relates to a targeted hydrophobic anti-tumor drug nanoformulation, which comprises a hydrophobic anti-tumor drug and a carrier with a mass ratio of 1:4-32.5, wherein the carrier is composed of 37.5-95.3 wt% albumin and 4.7-62.5 wt% hyaluronic acid-albumin conjugate, and the hyaluronic acid-albumin conjugate is prepared by albumin and hyaluronic acid with a molar ratio of 1:1-20. A preparation method of the nanoformulation comprises: preparing hyaluronic acid-albumin conjugate； formulating carrier solution； formulating drug solution； and preparing nanoformulation. The nanoformulation has even particle size distribution and good dispersibility, is stable without aggregating. The particle size of the lyophilized nanoformulation after being redissolved substantially remains unchanged, the yield of the nanoformulation is high and the efficacy is good after being filtered by a millipore filter (0.22 µm).
