The need for more cost-effective compounds is imperative because the demand for prebiotic compounds is ever on the rise. The focus of this study is the purification of the endoxylanase from B20 and its application in a cost-effective production of the prebiotic xylooligosaccharide (XOS) syrup having a high concentration of oligosaccharides. The extracellular endoxylanase was purified using ammonium sulphate fractionation, DEAE anion exchange, and Sephacryl gel filtration chromatography. The enzymatically produced XOS was used in the preparation of XOS syrup adopting the method of ultrafiltration with 10 and 3 kDa molecular weight cut-off (MWCO) membranes. Culture-dependent technique for the bacterial enumeration using selective probiotic microorganisms in an analysis was employed to confirm the prebiotic nature of XOS syrup. The molecular mass of the purified xylanase (XylB) was found to be approximately 85 kDa with the optimum pH and temperature of 6.5 and 60 °C, respectively. XylB hydrolyzed the xylan and produced short-chain xylooligosaccharides (XOS). At the end of the two-step ultrafiltration process, the hydrolysate was refi ned to form XOS syrup (44.4%) consisting of XOS with a degree of polymerization (DP) between 2 and 5, and >5. Among all the tested probiotic strains, exhibited maximum growth in the presence of 0.5% XOS syrup with a specific growth rate of 1.2 h. Through this study, we have identified a method to produce XOS syrup that can be used as an effective prebiotic supplement for the growth of several probiotic strains. Human gut probiotics was used as a model system for analysis of prebiotic oligosaccharide XOS, but for further confirmation of the prebiotic activity, feeding studies using animal models are needed to be carried out.