With the rapid increase of diabetes cases in the world, there is an increasing demand for slowing down and managing diabetes and its effects. It is considered that a viable prophylactic treatment for type 2 diabetes mellitus (T2DM) is to reduce carbohydrate digestibility by controlling the activities of α-amylase and α-glucosidase to control postprandial hyperglycemia and promote the growth of intestinal beneficial bacteria. In this work, the effects of sulfonated lignin with different sulfonation degrees (0.8 mmol/g, SL1; 2.9 mmol/g, SL2) on the inhibition of α-amylase and α-glucosidase and the proliferation of intestinal beneficial bacteria in vitro were investigated. The results showed that both SL1 and SL2 can inhibit the activity of α-amylase and α-glucosidase. The inhibition capacity (IC50, 32.35 μg/mL) of SL2 with a low concentration (0-0.5 mg/mL) to α-amylase was close to that of acarbose to α-amylase (IC50, 27.33 μg/mL). Compared with the control groups, the bacterial cell concentrations of Bifidobacteria adolescentis and Lactobacillus acidophilus cultured with SL1 and SL2 increased in varying degrees (8-36%), and the produced short-chain fatty acids were about 1.2 times higher. This work demonstrates the prospect of sulfonated lignin as a prebiotic for the prevention and treatment of T2DM, which provides new insights for opening up a brand new field of lignin.