We have established an orientation technique of microtubules and evaluated their polarities by the movement of kinesin-coated beads quantitatively in poly(dimethyl siloxane) (PDMS) channels. More than 95% of beads moved to the desired direction; this indicates almost all the microtubules were functionally oriented including plus and minus polarities. The technique is essential for fabricating a bio-hybrid nanotransport system, in which the kinesin−microtubule system combined with microfluidic structures provides a driving mechanism in an aqueous environment:  once microtubules are immobilized inside a microfluidic channel, kinesin-coated objects can be transported on the designated pathways without any liquid manipulation.