Liposomes have shown to be an excellent drug delivery system, but the short in vivo fate discourages their popularity. This work aimed to develop selenium-functionalized liposomes (SeLPs) for doxorubicin (Dox) delivery to prolong the systemic circulation of liposomes by in situ selenium coating and enhance the anticancer effect via the synergy between Dox and selenium. Dox-loaded SeLPs (Dox-SeLPs) were prepared by film hydration/active loading/in situ reduction technique and characterized by particle size, entrapment efficiency and morphology. The resulting Dox-SeLPs were 127 nm around in particle size (uncoated liposomes 107 nm) and were spherical in morphology. It was shown that Dox-SeLPs possessed a sustained release effect for Dox and could increase the cellular uptake of Dox compared with Dox-loaded liposomes (Dox-LPs). The accumulative Dox release from Dox-SeLPs was 46.5% and it was 64.9% for Dox-LPs within 84 h. Moreover, Dox-SeLPs exhibited slower drug release in the fetal bovine serum. Trafficking pathway study revealed that clathrin-mediated endocytosis and macropinocytosis were involved in the cellular uptake process of Dox-SeLPs. The in vitro cytotoxicity and apoptosis test indicated that Dox-SeLPs had higher cytotoxicity than that of free Dox and Dox-LPs. Dox-SeLPs showed a IC50 of 0.92 ± 0.16 μg/mL on A549 cells, far lower than that of free Dox (4.40 ± 0.58 μg/mL) and Dox-LPs (5.68 ± 0.73 μg/mL). Dox-SeLPs significantly improved the pharmacokinetic property and enhanced the antitumor efficacy of Dox in tumor-bearing mice. In conclusion, SeLPs exhibit good sustained release for Dox and have synergic anticancer effect with Dox, which may be promising as drug delivery vehicle.