Nature's biomaterials such as peptides and proteins represent a valuable source of highly defined macromolecules. Herein we developed a nanoparticle drug delivery system based on the assembly of surface-modified proteins that can be transferred into organic solvents and represent the structural material of the carrier system. The particles are prepared by an oil-in-water nanoemulsion technique without the need of additional denaturation or cross-linking steps for stabilization. We achieve the necessary lipophilic solubility switch of the protein material by high surface PEGylation under conservation of the native three-dimensional protein structure. This study focuses on lysozyme as model enzyme for the preparation of empty and doxorubicin-loaded nanoparticles with an average diameter of 100 nm. The particles are stable in physiological buffers and only release their therapeutic payload into cancer cells after a time-dependent cellular uptake. We also transferred this approach to various proteins, exemplifying the universal applicability of our new preparation method for protein-based nanoparticles.