Aims: This study focuses on the development of a new strategy xylooligosaccharide (XOS) production from aqueous ammonia-pretreated rice straw (A-PRS), followed by beta-xylosidase hydrolysis produced by the newly identified strain Weissella cibaria FB069. Methods and Results: We report a higher efficiency of A-PRS, including the removal of lignin and increase in cellulose and xylan content, compared to that of the alkali and stream explosion methods. Using the ammonia pretreatment method, rice straw was used to obtain 32.4% xylan. The crude xylanase from W. cibaria was used to hydrolyse A-PRS over different hydrolysis times. The highest XOS yield (131 mg XOS per gram rice straw) was observed after 10 h. XOS produced from the PRS was tested on stimulation effect on Bifidobacterium and Lactobacillus. Conclusion: The possibility of XOS production from PRS using beta-xylosidase with strong prebiotic properties. Significance and Impact of the Study: We investigated the new strain for signification production of XOS. The two-stage process here described could help to further explore the optimization conditions for prebiotic production. Additionally, the stimulation effect of XOS from alternative source has a promising prospect in functional food.