A microfluidic technique was developed to pattern nanoparticle monolayer controllably in a tentative polydimethylsiloxane (PDMS) microchannel. It was found that nanoparticle monolayer could be achieved in a twostep fluidic process: nanoparticle sedimentation and DI water rinsing. When nanoparticle suspension flows through a tentative PDMS microchannel, the particles will settle down due to the gravity effect and the Brownian motion and be captured onto the amino-functionalized substrate via electrostatic attraction. Aggregation on the substrate followed by a necessary DI water rinsing transforms hilllike nanoparticle aggregates into monolayer. Removing the tentative PDMS microchannel, pattern of nanoparticle monolayer following the channel shape was obtained. Experimental results indicated that the final monolayered nanoparticle coverage decreases when the flowing velocity in the sedimentation and/or the rinsing steps increases. Based on the continuity essence of fluid flow, different flowing velocities could be realized in one microchannel by varying the channel size. Therefore, monolayer patterns with controllable coverage could be achieved by carefully designing the microchannel width. The present approach is believed to be a promising nanoparticle monolayer patterning technique.