Abstract: The effect of metal ions (i.e., Ca2+, Cu2+ and Zn2+) on the interaction between folic acid (FA) and bovine serum albumin (BSA) under physiological condition was studied by fluorospectrophotometry and UV-spectrophotometry. It was shown that the inherent fluorescence of BSA was quenched in the presence of either the folic acid or the 3 metal ions. Judging from the fluorescence quenching constants obtained by Stern-Volmer equation, fluorescence quenching of BSA by FA, Ca2+, Cu2+ and Zn2+ were attributed to static quenching. Values of thermodynamic parameters ΔS and ΔG were calculated and based on these results, the binding force between FA and BSA was recognized to be electrostatic attraction in the absence of metal ions; while in the presence of Ca2+, the main binding force between FA and BSA was hydrogen bond and Van der Waals force; and in the presence of Cu2+or Zn2+, the binding force between FA and BSA was due to the hydrophobic effect. The binding constants between FA and BSA were changed in the presence of metal ions, but number of binding sites was still kept at around 1.