This study presents a direct fabrication process of nanofiber scaffolds within a pillar-based microfluidic device by using electrospinning and picosecond laser pulses. The picosecond laser irradiation source is a type of ultrafast laser, which facilitates adequately controlled processing in a glass microfluidic device at a wavelength of 355 nm. The required nanofiber scaffolds can be formed through electrospinning with an optimal maximum pore area of 8.4 mu m(2) at a concentration of 11 wt%. Subsequently, this study presents three types of pillar structures with nanofiber scaffolds in the microfluidic device. Self-organization of nanofibers can be achieved within the microfluidic device because of the incline structures. The nanofiber scaffolds were observed to fill the space between the pillars, including the side wall and bottom layer of the pillars and microfluidic device. This micromachining technique can be employed to fabricate downscaling patterned device structures for microfluidic applications. (C) 2017 Elsevier B.V. All rights reserved.