Design, simulation, flow characteristics of micro check valves used in Interstitial Fluid (ISF) devices are presented in this paper. The micro check valve includes three layers: the upper valve seat, the lower valve seat and the middle membrane layer which is fabricated by a polymeric surface micro machining process, which utilizes SU-8 as the functional material. The structure of membrane is designed as a disc with 1.2 mm diameter suspended on 4 folded beams, which work as valve springs. Maximum deformation and stress of membrane (50 μm, 100 μm, 150 μm, 200 μm, and 250 μm thicknesses) have been analyzed with ANSYS under 70 kPa. Simulation results reveal the membrane size is how to affect the performance of it. Finally, the membrane is fabricated with 150 μm SU-8 layer, and it is successfully integrated into the ISF sampling device. The forward flow and cut-off performance of the micro check valves have been observed with water. The flow curves were obtained under different forward and backward pressure. Experiment results shown that flow rates in the forward direction is small, and the flow rate is about 450 μL/min under 70 kPa; the valves can withstand more than 83 kPa pressure, and the reverse flow is nearly to zero, which meets the requirements of the ISF sampling device well.