There is increased demand for nanoparticles with a high fluorescence yield that have the desired excitation wavelength, surface functionalization, and particle size to act as biological probes. Here, a simple, rapid, and robust method, Flash NanoPrecipitation (FNP), to produce such fluorescent nanoparticles is described. This process involves encapsulation of a hydrophobic fluorophore with an amphiphilic biocompatible diblock copolymer in a kinetically frozen state. FNP is used to produce nanoparticles ranging from 30 to 800 nm with fluorescence emission peaks ranging from, but not limited to, 370 nm to 720 nm. Such fluorescent nanoparticles remain stable in aqueous solutions, and, in contrast to soluble dyes, show no photobleaching. Fluorophores and drugs are incorporated into a single nanoparticle, allowing for simultaneous drug delivery and biological imaging. In addition, functionalization of nanoparticle surfaces with disease‐specific ligands permits precise cell targeting. These features make FNP‐produced fluorescent nanoparticles highly desirable for various biological applications.

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