This paper presents a novel method of three-dimensional fabrication using Mask-less exposure equipment and a three dimensional microfluidic application for the cell manipulation. The grayscale data can directly control the height of the photoresist to be exposed without using any mask. Three-dimensional microchannel was successfully fabricated simply by using the low cost exposure system with the height range of 0-200 μm. We have succeeded in removing the zona pellucida of oocyte passing through the 3D-microchannel whose cross section is gradually restricted along the path to provide mechanical stimuli on the surface of the oocyte in every direction. This microfluidic chip contributes to the effective high throughput of the peeled oocyte without damaging them.