A thermal-responsive polymer was prepared by partially acetalyzing poly(vinyl alcohol) (PVA). The completely reversible polymer aggregation and dissolution occur above and below a low critical solution temperature (LCST) for the aqueous solution of the modified PVA. The partially acetalized PVA (APVA) with higher molecular weight and higher degree of acetalysis exhibited a lower LCST transition and was used as an anionic polymer for polymer complexation. Water-soluble polymer, cationic polyhexamethylene guanidine hydrochloride (CPHGH) with antimicrobial property, was also prepared. In conjunction with APVA, CPHGH created the unique antimicrobial polymer multilayers on the surfaces of rayon fibres via layer by layer (LbL) assembly. AFM images revealed that the particles generated by multilayers became larger after the material was treated at 60°C; while the roughness of the surfaces was increased as the layer number increased and then decreased. Moreover, antimicrobial tests also demonstrated that the rayon fiber assembled with (CPHGH/APVA) multilayers exhibited higher antimicrobial activity against E. coli and s. aureus.