Nanoparticles (NPs) have been widely studied and shown significant potential in biological and medical applications owing to their unique physicochemical properties. However, aggregation will severely restrict NP use by causing a change in biological effects of related NPs. This study experimentally investigated the aggregation behaviors of AuNPs in deionized (DI) water and whole blood by transmission electron microscopy and dynamic light scattering. The effects of NP size, NP concentration, temperature, and surface modification were analyzed and experimental results were rationalized by the extended Derjaguin-Landau-Verwey-Overbeek theory. The results show the advantage of selecting relatively large NP size and low NP concentration within the applicable range in achieving good dispersion. Higher temperature can enhance NP aggregation both in terms of aggregation rate and in aggregate size. In whole blood, the percentage of the AuNPs over 200 nm increased by more than threefold from 21 °C to 57 °C. Compared with the NP size and concentration, temperature most significantly affected NP aggregation. NP concentration exerts a relatively slighter influence than temperature and NP size when it changes within a relatively narrow range. The degree of NP aggregation in whole blood is higher than that in DI water as the ions and proteins will neutralize the electric charge and decrease the electrostatic force between NPs. Hydrophilic surface modification can effectively inhibit NP aggregation by increasing the electrostatic force, weakening the hydrophobic force, and preventing the Au-protein assembly. However, NP aggregation of hydrophilic polyethylene glycol-modified AuNPs was still observed in whole blood.