<p>Electrospun nanofiber mats are promising materials for capillary-driven microfluidic devices because their nanoscale inter-fiber spaces can generate strong capillary forces. In this study, we fabricated capillary-driven microfluidic transport systems using electrospun nanofiber mats with different degrees of fiber alignment and investigated the effect of fiber alignment on capillary-driven liquid transport and the minimum solution volume required for electrochemical sensing by cyclic voltammetry (CV). Fully hydrolyzed poly(vinyl alcohol) (PVA) was used as the nanofiber material, and annealing treatment was applied to impart water resistance. Fiber alignment was controlled by adjusting the rotational speed of the collector and the nozzle-to-collector distance during electrospinning (ES). The minimum measurable solution volume was reduced to 3 µL for the aligned nanofiber mats, whereas 4 µL was required for the randomly oriented mats. Based on the Lucas–Washburn framework, an effective capillary transport coefficient (<i>K</i> = <i>h</i>²/<i>t)</i> was introduced to quantitatively discuss the transport behavior. The largest enhancement was observed for the aligned nanofiber mat prepared at 1200&#xa0;rpm and annealed at 160&#xa0;°C, which exhibited a 4.4-fold higher <i>K</i> value than the randomly oriented mats prepared at 500&#xa0;rpm.</p>