Synthetic polymer single-chain nanoparticles (SCNPs) are an emerging new class of nanomaterials that possess similar folded structures as natural proteins. However, most SCNPs reported so far are packed loosely in their interior, resembling those of intrinsically disordered proteins (IDPs). Here, we report a facile strategy to synthesize SCNPs with controllable folding in aqueous solution. The precursor was mainly composed of N-isopropylacrylamide (NIPAM), a small percentage of hydrophobic photo-cross-linkers, and their hydrophilic counterparts. Contrary to conventional approaches that started from extended self-avoiding chain conformations, we started from prefolded conformations of thermoresponsive precursors at different temperatures. SCNPs were synthesized in situ by triggering reactions between hydrophobic photo-cross-linkers located in the interior under UV light irradiation. Folding structures ranging from loosely packed IDP-like to fully collapsed globules were obtained at different temperatures. Our experimental findings were also confirmed by using molecular dynamics simulations. Theoretical scaling analysis of the hydrodynamic radius Rh and molecular weight distributions for both precursors and corresponding SCNPs showed that these SCNPs synthesized at high temperatures (>305 K) indeed had fully folded globule structure. Besides, these SCNPs were thermoresponsive in a wide range, and near human body temperature; thus, could be superior candidates for future applications in drug cargo systems.