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When treating a composite mustard tuber wastewater with high concentrations of salt (about 20 g Cl⁻ L⁻¹) and organics (about 8000 mg L⁻¹ COD) by an anaerobic sequencing batch biofilm reactor (ASBBR) in winter, both high salinity and low temperature will inhibit the activity of anaerobic microorganisms and lead to low treatment efficiency. To solve this problem, betaine was added to the influent to improve the activity of the anaerobic sludge, and an experimental study was carried to investigate the influence of betaine on treating high salinity mustard tuber wastewater by the ASBBR. The results show that, when using anaerobic acclimated sludge in the ASBBR, and controlling biofilm density at 50% and water temperature at 8–12 °C, the treatment efficiency of the reactor could be improved by adding the betaine at different concentrations. The efficiency reached the highest when the optimal dosage of betaine was 0.5 mmol L⁻¹. The average effluent COD, after stable acclimation, was 4461 mg L⁻¹. Relative to ASBBR without adding betaine, the activity of the sludge increased significantly. Meanwhile, the dehydrogenase activity of anaerobic microorganisms and the COD removal efficiency were increased by 18.6% and 18.1%, respectively.