This study described an in-vitro wound-healing analytical system composed of a micro automated scratcher and an oxygen gradient micro-chamber. Cell migration assay using sharps has been used for investigating wound healing process, tumor metastasis, and angiogenesis formation. However, the technique is unable to control the physical size of scratching, because scratching is created manually by handy sharps, such as a pipette yet. For obtaining scratches having a contact physical dimension, this study attempted to develop a highly accurate cell-scratching instrument and demonstrated more efficient and precise physiologically investigation on cell migration in a gas gradient micro-chamber to be performed. A micro automated scratcher, composed of X, Y, Z, and θ-axes linear actuators, a pressure sensor, and a micro comb fabricated by a three-dimensional (3D) printer, was developed. Actuators controlled X, Y, Z, and θ directions, and a pressure sensor detected the contact of comb and the bottom of cell culture chamber. This scratcher was able to create of scratches with a 150 μm-scale width at a standard deviation of less than 13%. This high controllability and reproducibility were able to provide a well-controlled cell scratching in an oxygen gas gradient chamber, which allowed cell migration to be analyzed under various oxygen tensions (0-150mmHg) working as a function of wound healing process in one test. As a result, a hypoxia condition from 4 to 10 mmHg was found to promote the closing of cell scratching caused by cell migrations. The proposed scratching system, which had a precise cell-scratching capability and an oxygen gradient micro device, would offer an efficient experimental platform for the future cell migration studies, which would contribute to drug screening, tumor translational research, and regenerative medicine.