This paper describes a technique of characterizing microfluidic flow profiles from slow laminar flow to fast near-turbulent flow. Using a photo-activated fluorophore, nanosecond-duration photolysis pulses from a Nitrogen laser, and high-sensitivity single-molecule detection with Ar+ laser excitation, we report the measurement of flow speeds up to 47 m/s in a 33-microm-wide straight channel and the mapping of flow profiles in a 55-microm-wide microchamber. Sensitive single-molecule detection is necessary both because of the short time delay (submicrosecond) between laser photolysis and fluorescence detection and the fast transit times (as low as 10 ns) of the fluorescent molecules across the diffraction-limited beam waist of the Ar+ laser focus. This technique permits the high-resolution three-dimensional mapping and analysis of a wide range of velocity profiles in confined spaces that measure a few micrometers in dimension.