Gold nanoparticles (Au NPs) have a promising future in the diagnosis and therapy of diseases owing to their unique photoelectric properties. In the body, monodisperse Au NPs may aggregate extracellularly and intracellularly, which influences their in vivo fate and physiological effects. However, due to the lack of a rapid, precise, and high throughput method for characterizing Au NP aggregates, the intricate aggregation process of Au NPs has not yet been fully understood. In order to overcome this obstacle, we develop a single-particle hyperspectral imaging method to identify Au NP aggregates, making use of the outstanding plasmonic properties of monodisperse and aggregated Au NPs. This method enables us to monitor the dynamic formation of Au NP aggregates in biological mediums and cells. Further single-particle hyperspectral imaging analysis reveals that following exposure to 100 nm Au NPs, the formation of Au NP aggregates in macrophages highly depends on exposure dosage and is less affected by exposure duration. In particular, surface coating, including PEGylation and protein corona, can considerably mitigate the intracellular aggregation of Au NPs. Our findings highlight that single-particle hyperspectral imaging is an efficient way for studying how Au NPs aggregate in biological scenarios.