Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems.