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Gaussian distribution was used to describe the rough surface, and a novel method was proposed to generate 3-D rectangular rough microchannel by using Coons surfaces. With this method, one smooth microchannel and three rough microchannels were created. Constant wall temperature condition was applied to simulate the laminar flow of the saturated water in microchannels, and the effects of the surface roughness on flow and heat transfer characteristics were all investigated. The results indicated that different from the smooth microchannel, Poiseuille number and average Nusselt number were larger than the theoretical values, and increase approximately linearly with Re. As the height of the surface profile changed randomly, both pressure drop and local Nusselt number along the channel fluctuated randomly. The larger the roughness was, the more intense the fluctuation was. Poiseuille number and average Nusselt number increased with increasing equivalent relative roughness. Furthermore, within the scope of the Re, the effect of relative roughness on laminar heat transfer was stronger than that on flow resistance. In the microchannel, isotherm migrated to the lower surface, where the roughness was larger. As a result, the temperature distribution was asymmetric. As the gap of the roughness got increasingly larger, the asymmetry was more and more distinct.