C₆₀, as one of carbon nanomaterials widely used in various fields, could be released into the water environment thus exerting some potential health risks to human beings. This work examined the behavior of aqueous stable colloidal nano-C₆₀ (nC₆₀) aggregates under different environmental conditions including Polyethylene glycol octylphenol ether (TX100) micelles concentration, pH, and reaction time when exposed to TX100 micelles. Results show that the nC₆₀ aggregates became more dispersive and restored the capability of generating the singlet oxygen when exposed to TX100 micelles. With the increase of TX100 concentration, smaller average size of nC₆₀ aggregates was observed in dynamic light scattering (DLS) analysis, the fluorescence intensity of TX100 was more quenched by nC₆₀ aggregates, and the kinetic rate constant of generating the singlet oxygen for nC₆₀ aggregates was improved. The mean size of nC₆₀ aggregates in the presence of TX100 had no obvious variations when the pH ranged from 4 to 8. The longer reaction time between nC₆₀ aggregates and TX100 led to a higher kinetic rate constant of generating the singlet oxygen. Collective data suggest that variations in physicochemical properties of nC₆₀ aggregates are strongly dependent on the surrounding media under different environmental conditions and directly govern nC₆₀’s transport behavior and potential toxicity.