This paper describes a simple and reusable microfluidic SlipChip device for studying bacterial chemotaxis based on free interface diffusion. The device consists of two glass plates with reconfigurable microwells and ducts, which can set up 20 parallel chemotaxis units as duplicates. In each unit, three nanoliter microwells and connecting ducts were assembled for pipette loading of a chemoeffector solution, bacterial suspension, and 1X PBS buffer solution. By a simple slipping operation, three microwells were disconnected from other units and interconnected by the ducts, which allowed the formation of diffusion concentration gradients of the chemoeffector for inducing cell migration from the cell microwell towards the other two microwells. The migration of cells in the microwells was monitored and accurately counted to evaluate chemotaxis. Moreover, the migrated cells were easily collected by pipetting for further studies after a slip step to reconnect the chemoeffector microwells. The performance of the device was characterized by comparing chemotaxis of two Escherichia coli species, using aspartic acid as the attractant and nitrate sulfate as the repellent. It also enables the separation of bacterial species from a mixture, based on the difference of chemotactic abilities, and collection of the cells with strong chemotactic phenomena for further studies off the chip.