Convenient strategies to provide cell membrane-coated nanoparticles (CM-NPs) with multi-functionalities beyond the natural function of cell membranes would dramatically expand the application of this emerging class of nanomaterials. We have developed a facile approach to functionalize CM-NPs by chemically modifying live cell membranes prior to CM-NP fabrication using a bifunctional linker, succinimidyl-[(N-maleimidopropionamido)-polyethyleneglycol] ester (NHS-PEG-Maleimide). This method is particularly suitable to conjugate large bioactive molecules such as proteins on cell membranes as it establishes a strong anchorage and enable the control of linker length, a critical parameter for maximizing the function of anchored proteins. As a proof of concept, we show the conjugation of human recombinant hyaluronidase, PH20 (rHuPH20) on red blood cell (RBC) membranes and demonstrate that long linker (MW: 3400) is superior to short linker (MW: 425) for maintaining enzyme activity, while minimizing the changes to cell membranes. When the modified membranes were fabricated into RBC membrane-coated nanoparticles (RBCM-NPs), the conjugated rHuPH20 can assist NP diffusion more efficiently than free rHuPH20 in matrix-mimicking gels and the pericellular hyaluronic acid matrix of PC3 prostate cancer cells. After quenching the unreacted chemical groups with polyethylene glycol, we demonstrated that the rHuPH20 modification does not reduce the ultra-long blood circulation time of RBCM-NPs. Therefore, this surface engineering approach provides a platform to functionlize CM-NPs without sacrificing the natural function of cell membranes.