Zeolitic imidazolate framework-8 (ZIF-8) with porous structure, biocompatibility, and pH-sensitive release behavior is a promising nanoplatform for protein delivery. However, it is still a challenging task for a practical synthesis of protein-loaded ZIF-8 nanoparticles. Here we report an all-aqueous microfluidic reactor for one-step, rapid, and highly controlled synthesis of ZIF-8 nanoparticles with high protein loading at room temperature. Microfluidic reactor allows for an ultrafast (< 35 ms), complete mixing of Zn²⁺ ions and 2-methylimidazole (2-MIM) at different molecular ratios, leading to the formation of stable ZIF-8 nanoparticles with tunable sizes (13.2–191.4 nm) in less than 30 s. By pre-mixing various proteins such as bovine serum albumin (BSA) (isoelectric point (pI) = 5.82), ovalbumin (OVA) (pI = 4.82), or RNase A (pI = 8.93) with 2-MIM, ZIF-8 nanoparticles can be synthesized with protein encapsulation efficiency over 97%. Among the nanoparticles with different sizes, 25 nm ZIF-8 nanoparticles show the best performance in promoting the cellular uptake of protein payload. Using OVA as a model protein, we demonstrate that 25 nm ZIF-8 nanoparticles significantly enhance the cytosolic delivery of antigen, as indicated by the effective activation of dendritic cells. We anticipate that this microfluidic synthesis of nanomaterials may advance the emerging field of cytosolic protein delivery.