The influence of wall roughness on flow and heat transfer performance in microchannels at low Reynolds number is investigated in this paper. Two sizes of PMMA microchannels are fabricated by microinjection molding and the width is 20 μm and 800 μm respectively. The surface profile of bottom wall is described by the two-dimensional fractal geometry method and it is found there is error within 5% between surface roughness obtained by the fractal geometry method and actual roughness. Then, the effects of dimensionless relative roughness (5–7.5%), fractal dimension (1.5–1.8), aspect ratio (0.025–4) on the flow resistance and heat transfer performance are analyzed by numerical and experimental method respectively. Reynolds number considered here are 10–60. The results show that the better flow performance and heat transfer performance can be obtained with high aspect ratio of rectangular microchannel. However, increasing surface roughness not only increases the heat transfer performance, but also introduces a large flow resistance, which makes the friction coefficient rise sharply. As a result, surface roughness has great influence on the flow and heat transfer performance, and the most suitable surface morphology should be obtained according to the specific application.