The homogeneity of core–shell structure lipid-polymer hybrid nanoparticles (HNPs) is crucial for efficient intracellular cargo delivery. While microfluidic techniques have been recognized for their capabilities in precisely controlling the size and drug encapsulation efficiency, there is little research exploring the impact of mass transfer behavior within microchannels on the homogeneous assembly process of HNPs. Here, we manipulated the laminar flow state within TrM device to achieve controlled assembly of HNPs and explored a timescale dependent assembly technique to ensure the homogeneity of folate modified HNPs (FHNPs). Our founding indicate that this method enabled the control over the growth kinetics of PLGA cores within a timescale (τgrow) of 1–15 ms. The homogeneous ligand assembly on the surface of FHNPs can only be achieved when τgrow exceeds the timescale of functional lipid coating (τcoating). Compared to conventional bulk mixing methods, this strategy significantly reduces the risk of heterogeneous assembly in FHNPs fabrication and ensures consistent reproducibility across batches. The homogeneous FHNPs (HFHNPs) fabricated through this method exhibit precise control over particle size (ranging from 75 nm to 150 nm), low polydispersity index, and consistent core–shell structure. Furthermore, this strategy enhances the density of available folate ligands on the surface of FHNPs. In subsequent in vitro and in vivo anti-tumor assays, the icariside Ⅱ (IS) loaded HFHNPs (IS@HFHNPs) exhibited enhanced cellular uptake and tumor accumulation behavior. the timescale dependent homogeneous assembly method for HNPs developed in this research presents a promising avenue for maintaining quality in the synthesis of self-assembly composite nanomecidine.