At different temperatures, the spectral characteristics about the interaction between cefotaxime sodium and transferrin were analyzed by synchronous spectroscopy. Meanwhile, UV-absorption spectroscopy was used to supplement and verify the conclusion obtained from synchronous spectroscopy. Results showed that cefotaxime sodium quenched fluorescence from transferrin by the way of static quenching. New compound was generated by electrostatic force. The new compound did not stimulate or inhibit the interaction between subsequent ligand and transferrin. Tryptophan and tyrosine in transferrin both participated in the interaction. Circular dichroism spectroscopy displayed that Peptide chains of transferrin became loose and α-helical secondary structure of protein was changed.