Cell patterning has been widely used in research on fundamental cell biology and in applications such as tissue engineering, neuron network formation, cell based biosensor and drug screening. Although various methods have been developed, cell patterning in an enclosed microfluidic device at single cell level remains challenging. This paper describes a microfluidic device with microwells and protein patterns paired together in a single microchannel for an easy cell patterning. Cells captured in the microwells were positioned directly onto the protein patterns within 5 min and the patterning performance was successfully demonstrated using HeLa cells and human gallbladder carcinoma cells (SGC-996). Cells survived for 6 days in the microchannel. Cell attachment, migration, proliferation and cell colony formation were observed. Our device is free of topographic constraint for the patterned cells and no complex chemical modification to the substrate is needed, offering a simple, fast, and easy-to-operate way of patterning cells at single cell level in an enclosed microfluidic channel.