An optically active poly(diphenylacetylene) with a pendant amide functional group was synthesized by a macromolecular reaction of the optically inactive poly(diphenylacetylene) bearing carboxy groups with (S)-1-phenylethylamine. The obtained polymer showed good chiral recognition ability towards diverse racemates when used as a chiral stationary phase for high-performance liquid chromatography. The chiral recognition ability was substantially influenced by the chiral conformation, probably preferred-handed helical conformation, which was induced by thermal annealing in dimethylformamide after introducing optically active pendants through the macromolecular reaction.