Abstract: OBJECTIVE To carry out the optimization of formulation and preparation process of the transferrin and folic acid co-modified doxorubicin liposomes Tf (FA)-DOX-lipo, investigating the glioma targeting therapeutic effects. METHODS Tf (FA)-DOX-lipo was prepared by thin-film evaporation method and ammonium sulfate gradient method. First, folic acid was connected to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 to get the DSPE-PEG2000- Folic. Further, the phospholipid species, ratio of drug to lipid, hydration medium concentration and drug loading time were examined, particle size, encapsulation efficiency and stability were utilized to determine the formulation and process of the liposomes. Rats brain blood capillary endothelial cells (bEnd3) and astrocytes were used to construct in vitro blood-brain barrier(BBB), combined with rats glioma C6 cells to construct the composite BBB model, investigating glioma targeted therapy in vitro. The uptake mechanism of doxorubicin liposomes in bEnd3 cells, transport ratios through the BBB and toxicity on C6 glioma cells were investigated. RESULTS The results of NMR showed that folic acid was successfully connected to the DSPE-PEG2000-NH₂, while identifying DSPC as the main components of phospholipids, 120 mmol·L^-1 ammonium sulfate as the hydration medium, the drug to lipid ratio was 1∶15, drug time was 60 min, and stable dual-ligands modified liposomes with high encapsulation efficiency was successfully prepared. BEnd3 cell uptake was much larger than conventional liposomes (P<0.05), which demonstrated the brain targeting mechanism of Tf(FA)-dox-lipo were likely clathrin-dependent and caveolae dependent endocytosis pathway, subjected to the impact of transferrin and folic acid as well. In the BBB model, drugs transport ratio and toxicity on C6 cells after transport were significantly higher than other liposome groups. CONCLUSION Transferrin and folic acid modified doxorubicin liposomes have good glioma targeting and therapeutic effect in vitro.