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Nanoparticle (NP)‐based drug delivery systems or nanomedicines have broadened the horizon of translational research for decades. Conventional bulk mixing synthesis methods have impeded successful clinical translations of nanomedicines due to the limited ability of the controlled, scalable production with high uniformity. Herein, an on‐chip preparation of self‐assembled, drug‐encapsulated polymeric NPs is presented for their improved uniformity and homogeneity that results in enhanced anti‐cancer effect in vitro and in vivo. The NPs are formulated through rapid convective mixing of two aqueous solutions of a hydrophilic polymer and an anti‐cancer drug, doxorubicin (DOX), in the swirling microvortex reactor (SMR). Compared to conventional bulk‐mixed NPs (BMPs), the microvortex‐synthesized NPs (MVPs) exhibit narrower size distributions and better size tunability. It is found that the improved uniformity and homogeneity of the MVPs not only enhance cellular uptake and anti‐cancer effect with pH‐responsive drug release in vitro, but also result in an improved tumor regression and decreased side effects at off‐targeted organs in vivo. The findings demonstrate that uniformly designed NPs with more homogeneous properties can induce a significant enhancement of an anti‐cancer effect in vivo. The results show the potential of a high‐speed on‐chip synthesis as a scalable manufacturing platform for reliable clinical translations of nanomedicines.