Tire wear particles (TWPs) are commonly studied for their exudation toxicity, yet a critical knowledge gap exists regarding the source nature and migration of these particulate pollutants, hindering comprehensive environmental risk assessments. This study explores the pristine properties of three typical TWPs (rolling friction (R-TWPs), sliding friction (S-TWPs), and cryogenically milled tire treads (C-TWPs)) and their aging characteristics after incubation in runoff (primary aging) and sewage (further aging). Our investigation aims to unveil the intrinsic mechanisms of TWPs ecotoxicity towards freshwater biofilms. Results reveal that the generation modes significantly impact pristine physicochemical properties, including surface structure, particle size, and EPFR abundance. These factors, in turn, influence acute ecotoxicity, as evidenced by cell mortality, antioxidant enzyme activity responses, and metabolic changes in freshwater biofilms. The ecological toxicity ranking of pristine exposure groups is S-TWPs, R-TWPs, and C-TWPs, attributed to variations in surface properties and particle size. Following incubation and aging, especially in sewage, differences in physicochemical properties among TWPs types diminish. Alarmingly, ecotoxicity intensifies and becomes consistent across TWPs types, driven by the screening of small particles during water incubation aging and the formation of EPFRs on TWPs surfaces stimulated by photosensitive organic matter or groups. This study underscores the aquatic ecological risks associated with TWP surface properties, highlighting the significant influence of environmental aging conditions on these risks.