The present invention provides: a polysaccharide derivative containing a structure wherein hydrogen atoms in a hydroxyl group or amino group at the 2-position and the 3-position of a structure unit of polysaccharide are substituted with different substituents respectively represented by a specific general formula; and, a separating agent for optical isomers, which contains such a polysaccharide derivative. The present invention can provide a novel polysaccharide derivative which has excellent optical isomer separating ability, and is thus suitable for a separating agent for optical isomers, and can provide a separating agent for optical isomers which comprises the polysaccharide derivative.