Objective: This study was mainly focused on developing a dual-ligand liposomal delivery system to enhance both targeting specificity and cellular uptake.Methods: The specific ligand transferrin (TF) and the cationic cell-penetrating peptide TAT were connected with cholesterol via a polyethylene glycol (PEG) spacer to prepare the dual-ligand liposomes (TAT/TF-PEG-LP).Then the in vitro cellular uptake by three kinds of cells that possessed different expressing levels of transferrin receptor (TFR) was studied.The Ex vivo fluorescence imaging of tumors,the qualitative observation of tumor frozen section and the quantitative determination of cellular uptake in tumor tissues were used to evaluate the in vivo delivery efficiency of TAT/TF-PEG-LP.Results: The result of in vitro cellular uptake showed that TAT/TF-PEG-LP exhibited the enhanced cellular uptake and selectivity via the synergistic effect of both ligands in vitro compared to the single-ligand TAT or TF modified liposomes (TAT-PEG-LP or TF-PEG-LP),The ex vivo fluorescence imaging of tumors,the qualitative and quantitative evaluations of cellular uptake in tumor tissues altogether showed the in vivo delivery efficiency of TAT/TF-PEG-LP was higher than that of other liposomes.Conclusion: the dual-ligand liposomes co-modified with TF and TAT possessed a strong capability for synergistic targeted delivery of payload into tumor cells both in vitro and in vivo.