Doxorubicin (Dox) is a widely known chemotherapeutic drug that has been encapsulated into liposomes for clinical use, such as Doxil^® and Myocet^®. Both of these are prepared via remote loading methods, which require multistep procedures. Additionally, their antitumor efficacy is hindered due to the poor drug release from PEGylated liposomes in the tumor microenvironment. In this study, we aimed to develop doxorubicin-loaded lipid-based nanocarriers (LNC-Dox) based on electrostatic interaction using microfluidic technology. The resulting LNC-Dox showed high loading capacity, with a drug-to-lipid ratio (D/L ratio) greater than 0.2, and high efficacy of drug release in an acidic environment. Different lipid compositions were selected based on critical packing parameters and further studied to outline their effects on the physicochemical characteristics of LNC-Dox. Design of experiments was implemented for formulation optimization. The optimized LNC-Dox showed preferred release in acidic environments and better therapeutic efficacy compared to PEGylated liposomal Dox in vivo. Thus, this study provides a feasible approach to efficiently encapsulate doxorubicin into lipid-based nanocarriers fabricated by microfluidic rapid mixing.