In order to explore physicochemical and biological effects on detachment and activity recovery of aging biofilm by enzyme and surfactant treatments, two kinds of biofilm processes, i.e. biological aeration filter (BAF) and moving bed biofilm reactor ( MBBR), and multiple indicators including water quality, biofilm morphology, activity and microbial community structure, were employed. Results showed that detachment of aging biofilm was mainly attributed by extracellular polymeric substance (EPS) solubilization and dispersion, and activity recovery of aging biofilm mainly depended on biological effects of dominant bacteria. Phosphorus metabolism related bacteria, such as Microbacterium and Micropruina, were responsible for BAF biofilm regeneration. More abundant microbial community structure of MBBR regenerated biofilm was found, and biofilm activity was not only related to phosphorus metabolism related bacteria, but also to denitrifying bacteria. Rhamnolipid performed best on aging biofilm detachment and regeneration, giving a clue for effective activation of aging biofilm in wastewater treatment systems.