Nanoparticles (NPs) serve as a pivotal drug delivery system (DDS). However, preparing uniform NPs with targeted drug delivery has been challenging. By leveraging NP formulation with microfluidics and functionalization with an anticancer peptide (ACP), uniform drug-loaded NPs with tumor specificity can be obtained, leading to enhanced drug bioavailability, minimized side effects, and improved therapeutic efficacy. In this study, a microfluidic swirl mixer was used to prepare curcumin (Cur)-loaded PLGA NPs (Cur@PLGA NPs). Optimized formulation of Cur@PLGA NPs (90 nm with a PDI < 0.15) was obtained through precise regulation of process parameters. In addition, the cationic amphiphilic ACP CH(CH)CO-GIIKKIIKKI-NH (CG) was incorporated onto Cur@PLGA NPs (Cur@PLGA-CG NPs), resulting in an increased size of 155 nm with a reduced PDI of 0.07. The anticancer activity and cellular uptake of Cur@PLGA-CG NPs were comprehensively evaluated through 2D/3D in vitro experiments, revealing that the peptide-functionalized NPs exhibited specificity to cancer cells and demonstrated superior anticancer effects. Notably, Cur@PLGA-CG NPs demonstrated a rapid and effective cellular uptake in cancer cells. Moreover, these NPs demonstrated enhanced growth inhibition in 3D multicellular tumor spheroids. In vivo experiments further validated the efficacy of Cur@PLGA-CG NPs in controlling tumor growth rates in MCF-7 tumor-bearing mouse models without causing adverse effects on blood cells and vital organs. Overall, this drug delivery system provides robust support for the development of more effective drug delivery systems and treatment strategies in cancer therapy.