Extreme bleeding can make therapy challenging or even fatal. Even though several commercial homeostatic medicines have favorable clinical results, there is still a desire to discover materials that are effective, bio-safe, inexpensive, and simple to produce. In this study, regulated and uniformly sized chitosan microspheres (CSM) were fabricated using a simple process and assessed as hemostatic agents. In addition, the diameter criteria for optimal hemostatic effects were evaluated. Precipitation and coaxial airflow shear technique were used to fabricate CSM with a controlled and homogeneous particle size. Secondly, scanning electron microscope and N2 adsorption-desorption isotherm characterization, water absorption experiments, and in vitro coagulation evaluation demonstrated that the CSM produced in this experiment have a porous structure, a high specific surface area, and a high-water absorption capacity. The hemostatic effect was investigated using a rabbit blood coagulation experiment in vitro. To further examine the hemostatic effect using an arterial/venous bleeding model in rats. The results were superior to those in the blank control group. The hemostatic effect is related to the diameter of the microspheres. In these experiments, large diameter chitosan microspheres possessed superior hemostatic effect. Hence, these novel CSM show considerable potential for use in hemostatic events and demonstrated a diameter-dependent hemostatic effect.