The binding interactions of proteins with ligands play important roles in the physiological and biochemical processes of organisms. Thus it is a research hotspot in many fields. A key objective of the investigations on Protein-Ligand binding is to obtain their binding constant (K_b) for evaluating the strength of their binding. Fluorescence spectroscopy is widely used in Protein-Ligand binding studies as its high sensitivity, convenience, rapidity and low cost. When applying fluorescence spectroscopy, it is a key step to obtain the K_b value through mathematical analysis of fluorescence data with the aid of a functional equation. However, due to the different application range of different equations, the K_b values obtained by different equations for the same system are often varied. This work discussed the derivation process and the application conditions of the functional equations for calculating the K_b value of Protein-Ligand binding using fluorescence spectral data. Based on this, we summarized the optimal equation for the calculation of the K_b value for Protein-Ligand binding modes of 1:1 and 1:n (n≥2) under different preconditions. The discussion suggested that the selection of the optimal equation is based on two preconditions: a: Whether the Protein-Ligand complex is fluorescent; b: Whether the ligand concentration is much higher than the protein concentration. On the other hand, the human serum albumin (HSA)-Norfloxacin (NFX) binding system was used as a model, and the difference of the K_b values obtained by the fitting of different functional equations was compared and the corresponding reasons were analyzed. The results showed that HSA bound with NFX in 1:1 mode, and the K_b value was calculated to be 5.0×10⁴ L·mol^-1 at 298 K using the most appropriate equation (S12) for this system. However, the K_b values obtained by equation (S6) and (S24) were 28.8% and 48.6% higher than that by equation (S12) respectively, and the value obtained by equation (S17) was about two orders of magnitude higher. This intuitively showed the effect of choosing the inapplicable equation on the final result. In addition, the K_b value obtained by fluorescence spectroscopy in the present work was compared with those obtained by other techniques, indicating the relative reliability of fluorescence spectroscopy for the calculation of the K_b value in Protein-Ligand binding, and also pointing out the limitations of this method. This work warns us that when obtaining the K_b value of Protein-Ligand binding using fluorescence spectral data, it is very necessary to select the most suitable functional equation according to the preconditions (i.e. whether the hypothesis a and/or b are true or not), which will determine the reliability of the obtained results.