With the rapid science and technology advancement, the oil-water separation in oily wastewater has become an urgent problem, especially the emulsified oil-water mixtures. Hollow carbon spheres (HCSs) have tremendous potential in separating oil-water emulsions due to their rich porous channels and high surface-to-volume ratio. In this work, as-prepared chitosan/poly(γ-glutamic acid) nanoparticles crosslinked by Ni2+ (Ni2+/CS/γ-PGA NPs) were used as carbon precursor to fabricate HCSs. This strategy separated the formation process of the biomolecular microspheres and the carbonization process. Especially, the Ni2+/CS/γ-PGA NPs were fabricated from the self-assembly of chitosan and γ-PGA in aqueous solution and the crosslinking of Ni2+ via the electrostatic interactions, facilitating the formation of biomolecular microspheres and making the usable of biomolecule-based carbon precursors diversity. After lyophilization, Ni2+/CS/γ-PGA NPs powder was obtained, which was then carbonized in a tube furnace under N2 atmosphere. During the carbonization process, the nickel species aggregated together to form the core of nickel@carbon nanoparticles, and carbon formed the shell. At last, nickel nanoparticles were removed from the carbon framework by hydrochloric acid, obtaining HCSs with super-hydrophobicity and lipophilicity. The as-prepared HCSs exhibited excellent separation performance in oil-in-water emulsions.