The efficacy of CDK4/6 inhibitors as anti-tumor agents, especially in breast cancer, has been constrained by direct treatment-associated toxicities and the development of drug resistance. To overcome these limitations, we developed a novel lipid nanoparticle (LNP) platform utilizing microfluidic technology, a pioneering approach for the co-loading of small interfering RNA (siRNA) and hydroxychloroquine (HCQ). This innovative strategy leverages the synergistic effects of siRNA-mediated CDK4/6 silencing, which induces cell cycle inhibition, and HCQ-facilitated suppression of autophagy, enhancing anti-tumor therapy. Additionally, HCQ plays a pivotal role in improving the delivery efficiency of nucleic acid drugs by facilitating endosomal escape. In vitro studies demonstrated that co-delivery of siCDK4/6 and HCQ effectively blocked autophagy, arrested the cell cycle, induced cellular senescence, and significantly reduced tumor cell proliferation. Subsequent in vivo experiments confirmed the superior anti-tumor efficacy of this co-administration strategy compared to single-agent treatments, without observable adverse effects. This microfluidics-based LNP platform offers an engineerable strategy for the simultaneous delivery of small molecule drugs and nucleic acids, thereby providing immense potential for broader applications in cancer therapy.