This article explores implementation and application of fluorescence auto-correlation spectroscopy (FACS) as a quantitative immunoassay method. Using the binding interaction between Alexa Fluor647 fluorophore/green fluorescent protein antigen and its monoclonal antibody as a model system, we used a benchtop fluorescence correlation spectrometer to collect fluorescence autocorrelation data of solution samples of the antigen mixed with various concentrations of its antibody. Combining the classic FACS technique with the maximum entropy method, we can respectively evaluate antigen-antibody binding quantitatively and qualitatively. By analyzing the collected FACS data, the self-developed data analysis software can determine the dissociation constant of the antigen-antibody binding reaction. Experimental results show that FACS is a simple, rapid, and sensitive method for quantitatively determining antigen-antibody binding affinity, suitable for measuring dissociation constant values in the pM to nM range. The modular software standardizes the data analysis procedure while improving accuracy and reproducibility of analysis results.