Functionalized polystyrene (PS) microspheres are promising materials for the isolation of the chiral drugs due to their properties, such as a large surface area, high chemical stability and tunable morphology. Considering 10-100 μm microspheres are suitable as the matrix materials of the chiral stationary phase, and there are few reports on this. Here we report a facile synthesis of the tens-micron-sized carboxylated PS microspheres by a technology between the dispersion and suspension polymerization. The relation between the reaction solutions and the size of the carboxylated PS microspheres were investigated. The study showed that, the more H2O were used in the reaction solutions, which lead the principle of the reaction more close to the suspension polymerization, the larger size of the carboxylated PS microspheres. In addition, more acrylic acid would destroy the stability of the sphere morphology. Comparing the particle size distribution and morphology, it was revealed that tens-micron-sized carboxylated PS microspheres with narrow size distribution can be fabricated with the ratio of H2O/Ethanol 30/20 and the proportion of carboxyl groups reached to ~11%.