The interaction of Mn (II), Co(II) and HSA has been studied by fluorescence method at pH 7.4 and 5.3. Based on Forste non-radiative energy transfer theory, the distance between Trp-214 residue of HSA and the first strong binding site to Mn (II), Co (II) in HSA was determined, and it is much larger than that of reference. This notable different result has been discussed according to the binding site of Mn (II), Co (II ) and domain structure in HSA.