Fluorescence imaging is critical for physiological activities and cell biology but limited by the poor solubility, cell compatibility, and permeability. Herein, we develop a novel engineering methodology to prepare biocompatible and penetrable aggregation-induced emission (AIE) nanoparticles with the assistance of flash nanoprecipitation (FNP) technology. On the basis of the donor-π-acceptor (D-π-A) system, the AIE building block of tricyano-methylene-pyridine (TCM) is fine-tuned to long emission wavelength by modulating the π-conjugation bridge and electron-donating group, thereby achieving high solid fluorescent quantum yield, near-infrared (NIR) characteristic, large Stokes shift, and excellent photostability. On the basis of the FNP technology, the amphiphilic saponin solution and TCMN-5 in organic solvent are quickly mixed in the multi-inlet vortex mixer (MIVM), followed by saponin-encapsulation of the hydrophobic AIE nucleation with inhibiting further growth of nanoparticles. The biocompatible amphiphilic saponin such as α-hederin can encapsulate and micellize the AIE TCM fluorophore for efficient cell imaging. The kinetic FNP technology can not only modulate the uniform diameter size, but also distinctly increase the micelle stability when compared to the conventional thermodynamic self-aggregation method, which provides an alternative opportunity for scale-up preparation of drugs and probes in delivery vehicles.