Recent microfluidic advancements in oxygen gradients have greatly promoted controllable oxygen-sensitive cellular investigations at microscale resolution. However, multi-gradient integration in a single microfluidic device for tissue-mimicking cell investigation is not yet well established. In this study, we describe a method that can generate oxygen and chemical concentration gradients in a single microfluidic device via the formation of an oxygen gradient in a chamber and a chemical concentration gradient between adjacent chambers. The oxygen gradient dynamics were systematically investigated, and were quantitatively controlled using simple exchange between the aerial oxygen and the oxygen-free conditions in the gas-permeable polydimethylsiloxane channel. Meanwhile, the chemical gradient dynamics was generated using a special channel-branched device. For potential medical applications of the established oxygen and chemical concentration gradients, a tumor cell therapy assessment was performed using two antitumor drugs (tirapazamine and bleomycin) and two tumor cell lines (human lung adenocarcinoma A549 cells and human cervical carcinoma HeLa cells). The results of the proof-of-concept experiment indicate the dose-dependent antitumor effect of the drugs and hypoxia-induced cytotoxicity of tirapazamine. We demonstrate that the integration of oxygen and chemical concentration gradients in a single device can be applied to investigating oxygen- and chemical-sensitive cell events, which can also be valuable in the development of multi-gradient generating procedures and specific drug screening.