Soybean dregs fermented by Neurospora crassa is a typical traditional food in Gannan district of China. In this study, in vitro imitated gut fermentation was carried out to evaluate whether the oligosaccharides from this fermented soybean dregs had potential prebiotic properties. 11.91% of oligosaccharides were extracted from the fermented soybean dregs at the optimized condition which of 1:25 for ratio of soybean dregs (g) to 50% ethanol (ml), 90 min of extracted duration at 70 degrees C for twice. The soybean dreg oligosaccharides (SBOS) were progressively purified with Sevag method and on columns filled with AB-8 macroporous resin, and then identified as cellobiose by HPLC-ESI-MS and FT-IR. Oligosaccharides of soybean dregs with 800 mg/L significantly decreased pH value (p < 0.05) and ammonia N concentration (p < 0.05), and increased short chain fatty acid (SCFA) level (p < 0.05) in imitated gut fermentation compared with control group. It was shown that this fermented soybean dregs could be a potential prebiotic food.