Kinesin-driven microtubules have been a focus to serve as molecular shuttles to replace multiple on-chip functions in micro total analysis systems (μTAS). Although transport, concentration, and detection of target molecules have been demonstrated, controllability of transport directions is still a major challenge. To define multiple moving directions for selective molecular transport, we integrated the bottom-up molecular design of microtubules and the top-down design of a microfluidic device. The surface charge density and stiffness of microtubules were controlled, allowing us to create three different types of microtubules with different gliding directions corresponding to their electrical and mechanical properties. The measured curvature of gliding microtubules enabled us to optimize the size and design of the device for molecular sorting in a top-down approach. The integrated bottom-up and top-down design achieved separation of stiff microtubules from negatively-charged soft microtubules with approximately 80% efficiency under an electric field. Our method is the first to sort multiple microtubules by integrating molecular control and microfluidic device design, and is applicable to multiplexed molecular sorters.