This paper investigated the behaviors of flow and heat transfer of single-phase in rectangular micro-channels with three-dimensional numerical analysis. The single micro-channel is 200μm deep, 50μm wide. Deionized water was used as the working fluid. The fluid physical properties varying with temperature and Re number were studied. Comparisons were made among the results obtained from experiments, numerical simulations, and from those in the literature. The results indicated that the friction factors decreasing along the flow direction were ascribed to the fluid temperature rising under the unified heat flux boundary condition. It was found that influence of viscosity variation with temperature and viscous dissipation effect could be too significant to be neglected.