Endothelial cell monolayer (EM), acting as a barrier between blood and tissue, plays an important role in pathophysiological processes. Here we describe a novel microfluidic chip that is applied for convenient and high throughput in vitro permeability assays of EM. The chip included a gradient generator and an array of cell culture chambers. A microporous membrane as a scaffold component was built between a polydimethylsiloxane (PDMS) layer and a glass substrate to grow EM. Cell culture chambers were separated by microchannels and microvalves. The concentration gradient of compound solutions could be generated automatically and affected EM in different chambers. The permeability of EM at different time with histamine stimulation was in situ measured by the fluorescence detection of the leaked tracer. The existence of continuous flow in the channels allowed EM in a dynamic microenvironment and increased the amount of tracer through the EM, comparing to transwell assays. According to the prototype chip, the chip with a bigger array of cell culture chambers could be achieved easily and applied in the high throughput screening for drugs.