A novel use of confocal laser scanning microscopy (CLSM) makes the truly focused field-of-view with well-defined depthwise resolution possible for microscale particle image velocimetry (μ-PIV) applications. The operating principle of the CLSM is presented using the point spread function (PSF) that describes diffracted images of extremely small particles. The implemented high-speed CLSM system using a Nipkow rotating disk is applied to measure the microscale rotating Couette flow field confined between two parallel horizontal disks that are 180-μm apart, with the bottom one stationary and the top one rotating and seeded by 200-nm fluorescent spheres. The CLSM provides much distinct particle images in comparison with the conventional wide-field microscopy (WFM) and the measured vector profiles are more concentric and accurate depicting closer to an ideal Couette flow.