The reaction between carnitine and bovine serum albumin (BSA) in aqueous solution has been studied by fluorescence spectroscopy and absorbance spectra. The binding interaction between optical isomer, D-carnitine and L-carnitine, with BSA has been compared. Based on the site-binding model and fluorescence quenching, practical formulas for small molecular ligand binding to bio-macromolecule have been used, and the binding parameters were measured. The binding distance, the energy transfer efficiency between carnitine and BSA was also obtained by virtue of the Förster theory of non-radiative energy transfer. The effect of carnitine on the BSA conformation has been analyzed by using synchronous fluorescence spectroscopy. The influence of Fe3+ on the interactions between carnitine optical isomer and bovine serum albumin were also explored in this work. As a conclusion, molecular identification of BSA to carnitine isomer has been suggested preliminary.