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Alternative bio-refinery technologies are required to promote the commercial utilization of plant biomass components. The fructooligosaccharide (FOS) obtained after hydrolysis of the hemicellulose fractions was mainly applied in the pharmaceutical and food industries. Agricultural bi-product is a rich constituent in dietary fibres, which have prebiotic effects on the intestinal microbiota and the host. Herein we explored the impact of FOS on microbiota modulation and the gut homeostasis effect. High fructooligosaccharide recovery was obtained using alkaline extraction techniques. The enzymatic method produced fructooligosaccharides with minor contamination from fructan and glucan components, although it had a low yield. But combining the alkaline and enzymatic process provides a higher yield ratio and purity of fructooligosaccharides. The structure of the fructooligosaccharide was confirmed, according to FTIR, 13C NMR, 1H NMR and 2D-NMR data. Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods, which were found to be a cost-effective way to boost bio-refining value. Fructooligosaccharides with varying yields, purity, and structure can be obtained.