As the most representative of lignocellulosic materials, corn stalk (CS) will be a great candidate to produce xylo-oligosaccharides (XOS). Owing to the high impurity content of the XOS produced by directly enzymatic hydrolysis of xylan extracted from CS, subsequent refining steps are essential. The present study was aimed to investigate desorption during ethanol elution to improve the quality and antioxidant activity of XOS from CS. The desorption was systematically investigated after optimizing the elution conditions. The results showed that it had an elution watershed when the volume ratio was 2:1. More interestingly, XOS had a obvious priorities of desorption during ethanol gradient elution. The highest purity of XOS was 98.12% from 30% ethanol eluate. Antioxidant activity assay showed that the highest radical scavenging activity of XOS was 89.89% obtained from 70% ethanol eluate at a concentration of 3 mg/mL, which could be used in antioxidant food, feed additives.