This study proposed a Polydimethylsilcoxane (PDMS) concentration gradient microfluidic chip fabricated using MEMS, casting molding, and microimprinting technology. The chip is used to research on optimizing concentration of endothelial cells on glucose concentration gradient is extracted using this chip. In the experiment a concentration gradient in solution was generated by a microfluidic network, and the effect of concentration gradient in different flow rates were observed. Besides, it conjugates micropatterns that cell adhesion space width with 300 μm and gap with 200 μm to discuss the optimizing glucose concentration (0∼10%, 0∼20%, and 5∼15%) of endothelial cells. From the experimental data, we can prove that the concentration gradients are all stable under different flow rates (20, 30, 40, 50 μL/min). However, when flow rates reach over 20 μL/min, the cell would not remain on the substrate surface. The result shows that the glucose concentration gradient in the 8.66∼15% is the optimizing concentration of endothelial cells, and too high or low concentration will lead the cell to leave the substrate surface or to die. This study can solve the problems of the different drug concentrations during drug preparation and test. It will be used to drug concentration test and hypersensitive test in the future.