Algal-bacterial symbiosis (ABS) systems represent a highly promising approach for treating wastewater, yet how to establish ammonia-tolerant ABS systems through acclimation and the underlying tolerance mechanisms remain poorly understood. In this study, a novel acclimation strategy was developed to construct ammonia-tolerant ABS systems under high nitrogen stress. The acclimated system achieved a high ammonium removal efficiency of 75.8% at 1500 mg/L. Chlorophyll and extracellular polymeric substances (EPS) increased with ammonia levels, supplying energy for membrane synthesis. Tryptophan-like substances in loosely bound EPS enhanced membrane fluidity, while wrinkled microalgal surfaces expanded environmental contact. Fulvic acid-like substances promoted algal-bacterial interactions, evidenced by synchronous peaks at 500–1000 cm⁻¹ indicating systematic ion shielding and structural stability across the membrane-cell wall-hydration shell system. Metagenomic analysis revealed significant upregulation of ammonium assimilation, Calvin cycle, and phospholipid (phosphatidylserine and phosphatidylethanolamine) synthesis pathways. Ammonium incorporation into glutamate served as the primary nitrogen removal route. Microbial community profiling identifies a functional core dominated by microalgae, including Chlorella and Micractinium, as well as various bacteria such as Citrobacter and Microcella. Ecological network analysis suggested a cooperative mechanism whereby key bacteria (e.g., Citrobacter) facilitated non-dominant taxa (e.g., Hydrogenophaga), ultimately supporting microalgae as central players in ammonium degradation within the ABS system. In conclusion, this study elucidates the mechanistic basis of ammonia tolerance in ABS systems, emphasizing the critical roles of EPS-mediated protection, microalgae-driven ammonium assimilation, and cross-kingdom mutualism involving glutamate and cobalamin exchange.