Measurement of the water ﬂow rate in microchannel has been one of the hottest points in the applications of microfluidics, medical, biological, chemical analyses and so on. In this study, the scanning microscale particle image velocimetry (scanning micro-PIV) technique is used for the measurements of water flow rates in a straight microchannel of 200μm width and 60μm depth under the standard flow rates ranging from 2.481μL/min to 8.269μL/min. The main effort of this measurement technique is to obtain three-dimensional velocity distribution on the cross sections of microchannel by measuring velocities of the different fluid layers along the out-of-plane direction in the microchannel, so the water flow rates can be evaluated from the discrete surface integral of velocities on the cross section. At the same time, the three-dimensional velocity fields in the measured microchannel are simulated numerically using the FLUENT software in order to verify the velocity accuracy of measurement results. The results show that the experimental values of flow rates are well consistent to the standard flow rates input by the syringe pump and the compared results between numerical simulation and experiment are consistent fundamentally. This study indicates that the micro-flow rate evaluated from three-dimensional velocity by the scanning micro-PIV technique is a promising method for the micro-flow rate research.