Kinesin is a class of motor protein which moves on microtubules in eukaryotic cells. Because of its size and ability to convert ATP hydrolysis to mechanical force, engineering applications using kinesin as a novel power source in a nano-micro fluidic device have been of interest to wide range of researchers in nano-bio-technology field. However, the active transport driven by kinesins has not been well focused in conventional experiments. It is mainly caused by the fact that diffusional transport is also incorporated into the measurement of molecular transport in a conventional flow cell, which has 103-fold larger dimensions than that of molecules used in the kinesin-microtubule assay. In this paper, we designed a pneumatically-driven microfluidic device that confines the kinesin motility assay in channels, whose dimension is 2-µm wide and 1-µm height, for the evaluation of active transport by kinesin. Microtubules were successfully immobilized in channels and kinesin-driven transport of quantum dots (QDs) was observed. We measured the mass flux in microchannels by the active transport to compare a flux by diffusion at a given concentration gradient. We found that the kinesin-driven active transport was dominant at a concentration gradient lower than 10 nM/50 µm and comparable to axonal transport reported in vivo. These results are important knowledge toward the development of functional device driven by kinesin and in vitro axonal transport models.