Water-in-oil emulsion systems have recently attracted much attention in various fields. However, functionalization of water-in-oil emulsion systems, which is required for expanding their applications in industries and research, has been challenging. We now demonstrate the functionalization of a water-in-oil emulsion system by anchoring a target protein molecule. A microtubule (MT) associated motor protein kinesin-1 was successfully anchored to the inner surface of water-in-oil emulsion droplets by employing the specific interaction of nickel nitrilotriacetic acid histidine tag. The MTs exhibited a gliding motion on the kinesin-functionalized inner surface of the emulsion droplets, which confirmed the success of the functionalization of the water in-oil emulsion system. This result would be beneficial in exploring the roles of biomolecular motor systems in the cellular events that take place at the cell membrane and might also contribute to expanding the nanotechnological applications of biomolecular motors and water-in-oil emulsion systems in the future.