Abstract: Objective To develop a more reliable and stable method for determining monoclonal antibody (mAb) affinity constant based on competitive antibody/antigen binding. Methods The Kd value was calculated based on the relationship between the binding proportion of the antigen and the original concentration of the mAb that competed for the binding site of the antigen. Results The Kd values measured with this improved method under two different conditions were 2.61×10-12 mol/L and 2.39×10-12 mol/L, and those with Friguent method in these two conditions were 5.57×10-10 mol/L and 1.41×10-10 mol/L, respectively. Conclusion Compared with Friguent method, the Kd values measured with this improved method are closer to the actual value, and the measurement results under different experiment conditions are more stable.