Research on the origin, distribution, detection, identification, and quantification of polymer nanoparticles (NPs) in the environment and their possible impact on animal and human health is surging. For different types of studies in this field, well-defined reference materials or mimics are needed. While isolated reports on the preparation of such materials are available, a simple and broadly applicable method that allows for the production of different NP types with well-defined, tailorable characteristics is still missing. Here, we demonstrate that a confined impinging jet mixing process can be used to prepare colloidally stable NPs based on polystyrene, polyethylene, polypropylene, and poly(ethylene terephthalate) with diameters below < 100 nm. Different fluorophores were incorporated into the NPs, to allow their detection in complex environments. To demonstrate their utility and detectability, fluorescent NPs were exposed to J774A.1 macrophages and visualized using laser scanning microscopy. Furthermore, we modified the NPs in a postfabrication process and changed their shape from spherical to heterogeneous geometries, in order to mimic environmentally relevant morphologies. The methodology used here should be readily applicable to other polymers and payloads and thus a broad range of NPs that enable studies of their behavior, uptake, translocation, and biological end points in different systems.