To provide a method for simply separating and refining a neoagarotetraose (tetraose) and a neoagarohexaose (hexaose) from an agar oligosaccharide produced in production of the agar oligosaccharide by -agarase.

The method for separating and refining the agar oligosaccharide comprises selectively separating and refining the neoagarotetraose by dissolving a water-containing organic solvent into the agar oligosaccharide, separating the residue and drying the organic solvent, in a method for separating and refining the agar oligosaccharide produced by adding -agarase used as an agar degradative enzyme to agar used as a substrate.