Flash nanoprecipitation (FNP) is an easily scalable and fast processing method for the preparation of nanoparticles (NPs) with simple vortex equipment. By using the FNP method, fluorescent NPs are prepared in less than 1 s in a multi-inlet vortex mixer, in which hydrophobic aggregation-induced emission (AIE)-active dye of EDP is incorporated within the biocompatible block copolymer poly(ethylene glycol)-b-poly(epsilon-caprolactone) for EDP NP assembly. The formulation parameters of stream velocity, dyes, and loading and concentration in FNP are optimized. The sizes of the NPs ranged from 20 to 60 nm with a ratio change of mixed solvents. As a control, an aggregation-caused quenching (ACQ) molecule of BDP was also synthesized for BDP NPs. To gain insight into the effect of the polymer on the aggregation state of hydrophobic dyes, the preparation of EDP and BDP NPs without block copolymer was also investigated. Apparently, the sizes of the NPs display large distributions without an amphiphilic block copolymer as the engineering template, suggesting that the block of polymers plays a key role in tuning the aggregation state of encapsulated dyes in FNP processes. Moreover, the peak shifts of dye with different microenvironments also confirmed the successful encapsulation of fluorescent dye in the NP cores. Finally, by externally applied forces in the FNP method, the engineered assembly of AIE-active fluorescent NPs possessing a narrow size distribution with desirable fluorescence properties was obtained. These features provide the possibility of rapidly constructing controllable AIE-active fluorescent NPs as biomedical tracers.