Fluorescent microspheres (FMs) are tiny particles with special functions that are widely employed in biological research. Counting of microscale FMs is a great challenge by capillary electrophoresis. Herein we developed a method to count 2 μm FMs based on a microfluidic chip with a gradual change in inner size. Such a microfluidic chip can inhibit sample blocking at the inlet of the capillary. The results showed that FMs migrated in the wide part of the microchannel side by side, and then passed through the narrow part one by one. There was a linear relationship between the number of peaks in the electropherogram and concentration of FMs if they were running in the microchannel for more than 20 min. A high separation voltage may lead to aggregation of FMs in the microchannels, and about 2 × 104 FMs can be counted within 30 min by this microfluidic chip.