Gold nanoparticles (AuNPs) are widely used in biomedical applications, but much less is known about their immunological properties, particularly their interaction with the complement system, a key component of innate immunity serving as an indicator of their biocompatibility. Using a library of different-sized AuNPs (10, 20, 40, and 80 nm) passivated with polyethylene glycol (PEG) of different molecular weight (Mw = 1, 2, 5, and 10 kDa), we demonstrated that citrate-capped AuNPs activated the whole complement system in size-dependent manner, characterized by the formation of the end-point activation product, SC5b-9, in human serum. Although PEGylation of AuNPs mitigated, but not abolished, the activation level, complement activation by PEGylated AuNPs was independent of both the core size of AuNPs and molecular weight of PEG. The cellular uptake of both citrate-capped and PEGylated AuNPs by human U937 promonocytic cells which expresses complement receptors were highly correlated to the level of complement activation. Taken together, our results provided new insights on the innate complement activation by PEGylated AuNPs that are widely considered as inert biocompatible nanomaterials.