This study develops a scanning laser Doppler microscope (LDM) to measure the velocity flow profile within a capillary. The flow velocity is established by detecting the periodic fluctuation in the scattered light as the microspheres pass through a measurement volume with a diameter of 15 μm. Using a programmable positioning stage, the position of the capillary relative to the focal point is shifted incrementally in the x- and y-axis directions, respectively, such that the velocity flow profiles in the X-X′ and Y-Y′ directions can be obtained. During scanning, the required stage displacements in the x- and y-axis directions are calculated using an analytical model that compensates for deviations in the focal point position caused by the mismatched refractive index effect. The velocity–flow rate relationship within a capillary with an internal diameter of 75 μm is characterized and is found to be in good agreement with the theoretical prediction [correlation coefficient (R2): 0.998]. Additionally, the measured velocity flow profiles in the X-X′ and Y-Y′ directions are in good agreement with the analytical results obtained from the Navier-Stokes equation. The scanning LDM provides a powerful technique for characterizing microfluidic flow fields in the cross section of micro-electro-mechanical systems–based biochips and similar advanced applications.