Abstract: In this paper, bagasse was fermented to produce dietary fiber by Bacillus subtilis. The extraction process of soluble dietary fiber (SDF) was optimized by single factor test and response surface methodology. The physicochemical properties, structure and antioxidant activity in vitro of dietary fiber before and after fermentation were compared and analyzed. The results showed that the optimal extraction conditions were inoculum amount 10%, pH7 and fermentation time 71 h. Under these conditions, the extraction yield of soluble dietary fiber was 17.95%±0.06%. The analysis of physicochemical showed that the water-holding capacity, oil-holding capacity and swelling force of fermented dietary fiber (F-DF) were significantly increased compared with DF (P<0.05). The ultrastructure showed that the particle size of dietary fiber decreased and showed a lamellar state after fermentation. Fourier infrared spectrum showed that the intensity of dietary fiber absorption peak increased and the overall peak type and position did not change after fermentation. The X-ray diffraction pattern showed that the diffraction peak intensity decreased and the crystal structure did not change after fermentation. Compared with dietary fiber, the DPPH free radical scavenging energy, reducing power and hydroxyl radical scavenging rate of fermented dietary fiber were the highest, which were increased by 32.9%, 0.70 and 50.55% (P<0.05), respectively. The preparation of bagasse dietary fiber by fermentation could improve its physical and chemical properties and structure, and effectively improve its antioxidant activity. Therefore, this experiment provides a theoretical basis for the effective use of sugarcane raw materials, the processing and utilization of by-products and the avoidance of resource waste.