Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP–protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches.