Refolding a denatured protein into its native form and stabilizing its structure are significant in the production and storage of protein products. The self-assembled mixed-shell polymeric micelle (MSPM) based nanochaperones closely mimic natural molecular chaperones in terms of structure and function and have been widely used to regulate the (re)folding of denatured proteins. However, the traditional manufacturing method of MSPM based nanochaperones is inconvenient with a very low concentration, which greatly restricts its large-scale applicability in practice. Here, we developed a one-pot strategy to synthesize high-concentration MSPMs for the mass preparation of nanochaperones to refold bulk denatured proteins and stabilize the structure of native proteins. The high-concentration MSPM is synthesized using two macro-RAFT agents to induce the polymerization and cross-linking of hydrophobic monomers. Through this one pot synthesis method, MSPMs with a concentration of 36.8 mg ml(-1) are obtained, which is much higher than that obtained through the traditional method (0.5 mg ml(-1)). Moreover, the obtained MSPMs provide a good renaturation efficiency for the refolding of bulk denatured proteins and can stabilize native proteins for long-term storage. This strategy provides a simple method for the mass preparation of functional nanochaperones, which will promote its applications in practice.