The morphology, functions, and gene expression of blood cells and vessels, especially endothelial cells may be modulated by complex hemodynamic forces, as well as by chemical stimulation. Various models have been designed to simulate either the effects of blood flow or the chemical environment on these cells in an effort to understand their response. In this study, a double-layer flow chamber was developed to deliver different flow shear stress and to offer chemical factors, for mimicing the characteristics of both mechanic and chemical stimuli as in vivo conditions. The upper layer of the flow chamber is a main testing chamber (MTC), structured as a quadrate lumen, in which endothelial cells are seeded on the basal plane. The mechanical properties of cells exposed to flow shear stress can be recorded in real-time. The lower layer is a supplementary testing chamber (STC), which provides chemical stimuli. A partition microfiltration membrane exists between the two layers. The flow relationship among variables, such as the velocity, the shear stress, and the pressure distribution of the flowing in the chamber are achieved by using the nondimensional technique and the method of Bessel function of imaginary argument according to Navier-Stokes equations.