Five proline-derived acetylene monomers, (S)-N-aromatic carbamoyl-2-ethynyl pyrrolidine, were efficiently synthesized from the commercially available biomass-based starting material (S)-N-(tert-butoxycarbonyl)-prolinal. They were converted to the corresponding optically active helical polymers under the catalysis of a Rh-diene complex. These polymers exhibited excellent optical resolving power as the chiral stationary phases (CSPs) of high-performance liquid chromatography for hydrogen bond donating racemates and an organometallic complex. The correlations among the nature, size, and position of the substituent on the phenyl ring with the polymer conformation as well as the enantioseparation performance were systematically explored through NMR, Raman and UV-absorption spectroscopy and polarimetry, circular dichroism, HPLC, and computational simulation. It was found that the strength of the hydrogen bond and π–π interactions between enantiomers and CSPs played a remarkable role in enantioseparation. The strong electron-withdrawing substituent disfavored chiral recognition because of the reduced electron density of the ureal carbonyl group via the inductive effect, while the weak electron-withdrawing and electron-donating substituents favored optical resolution. The meta-substitution on the phenyl ring weakened the chiral discrimination on the large racemate. Moreover, reducing the eluent polarity tended to improve enantioseparation. This work helps explore the full potential of polyacetylene-based CSPs.