Cancer progression normally creates intrinsic hypoxic or oxygen gradients within tumor lesions that further instigate metastasis. Despite the advancements, recreating physiological oxygen gradients in vitro remains challenging yet a necessary requirement to develop effective point of care (POC) technologies for elucidating cellular migratory or therapeutic responses for personalized treatments. Here, we report a unique method of generating stable and linear oxygen gradients using the split/recombine strategy within microfluidic devices. Internal glass coating ensures proper stabilization of oxygen gradient and prevents gaseous escape. Underlying PtOEPK sensor enables accurate and instantaneous detection of gradients in real-time. The versatility of this gradient platform encompasses a wide variety of biological and biomedical analyses such as cell viability, cell migration, drug resistance, and ER (endoplasmic reticulum) stress responses under hypoxia and many of these are currently being explored.