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Herein, we present a new flow-guided assembly approach to align and position DNA nanowires. Single or multiple drops of a λ-DNA solution were loaded from one side of template with micropad array and blown off by air flow. DNA molecules were aligned along the flow direction and well positioned under the guiding of pre-defined micropatterns. Different from other pioneering work, we focused on investigating the assembly quality, reliability and appropriateness for large-scale manufacturing. We correlated the assembly quality (alignment, patterning and the surface coverage) with the concentration of DNA in droplets and droplet evaporation and suggested appropriate process windows. With this new approach, the synthesis, alignment and patterning of nanowires may be done in a reliable and efficient manner and with high throughput.