A novel amphiphilic polyisocyanate block copolymer with hydroxyl side groups was synthesized by a combination of living anionic polymerization and thiol-ene click chemistry. First, the living anionic block copolymerization of allyl isocyanate (AIC) and n-hexyl isocyanate (HIC) produced a well-defined block copolymer (PAIC-b-PHIC) as a precursor. The subsequent free-radical-mediated thiol-ene click reaction of this polymer with 2-mercaptoethanol at room temperature quantitatively converted the allyl side groups of the PAIC domain to hydroxyl groups, finally creating PAIC(OH)-b-PHIC. The amphiphilicity of PAIC(OH)-b-PHIC led to lamellar and cylindrical phase separations in the thin films cast from different solvents (THF and toluene). The functionalities and phase separation behaviors of PAIC(OH)-b-PHIC were characterized by NMR, SEC-MALLS, and TEM analysis.