The present invention relates to a method for producing marine algae-derived agarotriose, and a use thereof as a prebiotic. More specifically, the present invention investigates the characteristics of agarotriose as a prebiotic which is selectively metabolized by probiotic microorganisms, thereby enabling agarotriose to be used as an anti-cancer or anti-inflammatory agent in the fields of food and pharmaceuticals, and enabling agarotriose to be obtained at high yield through efficient purification with minimal loss after enzymatic hydrolysis of a red algae-derived polysaccharide without pre-treatment.