Bamboo powder (10.0 g) was subjected to autohydrolysis with a solid to liquid ratio of 1 : 10 under non-isothermal conditions to produce xylo-oligosaccharides (XOS) with a degree of polymerization of 2 to 6. The experiment was performed with two independent variables (reaction temperature 152–208 °C and reaction time 1.72–58.28 min) to optimize the reaction conditions by central composite design of the response surface methodology. Analysis of the variance of the regression model of XOS yield was in good agreement with the experimental results, and the predicted optimal condition for the production of xylo-oligosaccharides was observed at 182 °C for 31 min with a yield of 36.4%. Under the optimal reaction conditions for the production of XOS, relatively low concentrations of monosaccharides and byproducts were obtained. The investigation of antioxidant activity revealed that XOS produced from autohydrolysis exhibited a comparable scavenging activity to commercial antioxidants in superoxide and hydroxyl radicals.