We report the facile ambient temperature generation of size tunable and well-defined (pro)fluorescent single-chain nanoparticles (SCNPs) via the photoinduced nitrile imine intramolecular cross-ligation of linear precursor polymers, constituting a platform technology as novel imaging agents. A set of three linear precursor polymers (Mn ≈ 14000 g mol-1, Đ ≈ 1.25) was synthesized via nitroxide-mediated statistical copolymerization of styrene and 4-(chloromethyl)styrene (CMS), followed by a postpolymerization modification of the resulting copolymer installing protected maleimide (PG-Mal) as well as tetrazole (Tet) moieties. The tetrazole content (% Tet) along the lateral polymer chains was varied between 12 and 24% in order to preselect not only the size of the corresponding SCNPs, but also their fluorescence and reactive properties. Finally, the applicability of the profluorescent SCNPs for fluorescence labeling was demonstrated utilizing residual surface expressed Tet moieties on the SCNPs surface in a reaction with maleimide functional polymeric microspheres. The (pro)fluorescent single-chain nanoparticles were in-depth characterized by 1H NMR spectroscopy, dynamic light scattering (DLS), size exclusion chromatography (SEC), and atomic force microscopy (AFM), as well as UV/vis and fluorescence spectroscopy.