This paper presents the novel three-dimensional fabrication using maskless exposure equipment and threedimensional (3D) microfluidic cell manipulation uses grayscale data to directly control the exposed photoresist height without using a mask. The 3D microchannel and microvalve were fabricated simply using lowcost exposure and height ranging from 0 to 200 µm. The 3D microvalve prevents liquid leakage when the membrane is closed – difficult to do using conventional 2D photolithography. We removed the oocyte zona pellucida passing through the 3D microchannel whose cross-section is gradually restricted along the path to provide mechanical stimulation omnidirectionally on the oocyte surface. The microfluidic chip may contribute to make high peeled-oocyte throughput effective without damaging the oocytes.