Chiral molecularly imprinted polymer microspheres (MIPMs) reported so far are majorly limited to being constructed by using achiral polymer together with chiral template. The present contribution reports on a unique type of chiral MIPMs consisting of chirally helical substituted polyacetylene, which are prepared through suspension polymerization by using (a) chiral acetylenics as monomer and chiral Boc-D/L-proline as template. The resulting MIPMs after removing the template show optical activity that is derived from the chirally helical structures of substituted polyacetylene. The microspheres demonstrate enantio-differentiating ability in releasing the enantiopure templates. A complete release of the template provides the chiral MIPMs. Worthy to mention is that the two chiral sources (chirally helical conformation and chiral template configuration) work in a synergistic way, obviously increasing the MIPMs’ enantiodiscrimination ability. The present study develops a strategy for preparing chiral MIPMs, which are expected to find significant applications in chiral separation, enantioselective release of chiral drugs, etc.