Abstract: The reduction of nonspecific binding (NSB, or passivation) and functionalization of the diamond surface are crucial for nitrogen-vacancy (NV) center-based quantum sensing in biomedical systems, especially for biomolecular detection. In this work, we systematically investigated polyethylene glycol (PEG)-based treatment technologies for the passivation and functionalization of diamond surfaces. Specifically, we evaluated the passivation and functionalization effects of PEG with different chain lengths and then the impact of streptavidin proteins on these treatments. Our results show that either a mixture of 5 kDa PEG and biotin-PEG in a ≥ 5∶1 ratio (PEG to biotin-PEG) or 20 kDa biotin-PEG alone coordinated the suppression of NSB and efficient specific binding of biomolecules and nanoparticles. In addition, we confirmed that the magnetic-sensing-related properties of NV centers were not affected by the PEGylation process and verified the applicability of our diamond treatment scheme with an immunomagnetic assay of antibodies in human serum samples. Overall, our study provides an effective biocompatible treatment strategy and practical protocols for diamond surfaces, promoting NV center-based quantum sensing of biomolecules.