Abstract: In order to enhance the yield of oat non-starch polysaccharide (ONSP), the extraction process was optimized and its immunological activity was investigated. This study employed microbial fermentation-assisted extraction, optimizing the process for oat non-starch polysaccharide (ONSP1) using a one-way response surface methodology. Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) were employed to determine the structural characteristics of ONSP. Additionally, the immunological activity of ONSP was investigated and compared with that of oat non-starch polysaccharide (ONSP2) extracted through the conventional hot water method. The results demonstrated that the optimal extraction process for ONSP1 was achieved with a 5% of the inoculum and fermentation at 34 ℃ for 26 h, resulting in a yield of ONSP1 at 8.60%±0.04%. Both ONSP1 and ONSP2 were composed of glucose, arabinose, and xylose, with the contents of arabinose and xylose of ONSP1 were significantly higher than those of ONSP2 (P<0.05). Despite the lower molecular weight of ONSP1 (47.2 kDa) compared to ONSP2 (53.2 kDa), ONSP1 had a significantly higher β-glucan content (P<0.05). Furthermore, both ONSP1 and ONSP2 demonstrated the capacity to promote the proliferation of RAW264.7 cells, with ONSP1 exhibiting a more pronounced immune-stimulating activity. In conclusion, microbial fermentation enrichment resulted in oat non-starch polysaccharides with a higher β-glucan content and stronger immunostimulatory activity. This study provides new insights for further investigation into the bioimmunological activities and structural relationships of oat non-starch polysaccharides.