The compositional changes of extracellular polymeric substances (EPSs) in membrane bioreactors (MBRs) under different salinity shocks and their effects on membrane fouling, as well as the optimal design of operating parameters in high-salt environments have been analysed in this study. A gradient acclimation method was adopted to increase the salinity of the system to construct a 3.0% salinity MBR system. In the process of increasing the salinity from 0% to 3.0%, the degree of membrane fouling intensified, and the concentration of EPSs showed an inverted U-shaped change. Pearson's correlation analysis showed that SMPs are related to membrane fouling, polysaccharides and protein substances are the main contributors. In addition, by a response surface method, the membrane fouling rate (|K|) and ammonia nitrogen removal efficiency (R.E) were used as response values to obtain the optimization results of operating parameters, namely, HRT = 22.30 h, C/N = 4.80, DO = 1.5–2.0 mg L⁻¹, and the actual average values of |K| and R.E were 7.12 × 10¹² m h⁻¹ L⁻¹ and 85.18%, respectively. In general, research results provide guidance for the design of operating parameters in the MBR treatment of high-salt wastewater.