Background Calcium carbonate (CC) nanoparticles have broad biomedical utilizations, owing to their multiple intrinsic merits. However, bare CC nanoparticles do not allow for the development of multifunctional devices suitable for advanced drug delivery in cancer therapy. Methods Phospholipid-modified phospholipid–CC hybrid nanoparticles were prepared in our study using a combination of vapor-diffusion and solvent-diffusion methods to offer optimized pharmaceutical capabilities. Results Considering that particle size is a critical parameter that plays an important role in both in vitro and in vivo behaviors of nanoparticles, we here for the first time a present detailed protocol for the size-controlled preparation of hybrid nanoparticles, as well as analysis of the in vitro/in vivo behaviors of differently sized hybrid nanoparticles. Conclusion Our results might significantly advance the application of this promising material in more varied fields.