The invention discloses a preparation method of substituted acetylene helical polymer microspheres, and belongs to the field of high-molecular material. The preparation method comprises following steps: firstly, polyvinyl alcohol (PVA) is dissolved in deionized water so as to prepare a polyvinyl alcohol aqueous solution; substituted acetylene monomer is dissolved in dichloromethane, trichloromethane, or methylbenzene, and a rhodium metal catalyst is added, and an obtained mixture is stirred for 5 to 30min, and is added into the polyvinyl alcohol aqueous solution dropwise under nitrogen protection; an obtained mixed material is subjected to mechanical stirring, is reacted for 1 to 24h at a temperature of 0 to 60 DEG C, is filtered after reaction, is washed with deionized water and ethanol successively, and at last is subjected to vacuum drying for 12 to 24h so as to obtain yellow microspheres. According to the preparation method, the substituted acetylene helical polymer microspheres are prepared via suspension polymerization, application range of substituted acetylene helical polymer is enlarged, and the substituted acetylene helical polymer microspheres possess recoverability, and can be reused.