The interaction between ferrocene derivatives, such as Fc(COOH)2(λmax=286 nm), Fc(OBt)2(λmax=305 nm), Fc(Cys)(λmax=289 nm) and heme(λmax=386 nm) were studied by UV-Vis spectroscopy, respectively. The results show that, when the concentration of heme is fixed, the absorbance of heme increases with the increase of Fc(COOH)2 and Fc(Cys) concentration, the absorbance of heme almost keep the same when Fc(OBt)2 concentration increases; when the concentration of ferrocene derivatives are fixed, the absorbance of Fc(COOH)2 and Fc(Cys) also increases with the increase of heme concentration, the absorbance of Fc(OBt)2 almost keep the same when heme concentration increase. It is demonstrated that the hydrogen bonding interactions happen between Fc(COOH)2, Fc(Cys) and heme, none of Fc(OBt)2, the formation of hydrogen bonding lead to the growth of molecular chain, the bigger molecule can absorb more energy and increase the absorbance. Meanwhile, the stability of molecule is affected by the formation of hydrogen bonding, when the reaction time increases from 0.5 h to 18 h and 48 h, the absorbance at λmax=384 nm change from 2.64 to 2.53 and 2.51 with fixed concentration of Fc(COOH)2, the absorbance at λmax=384 nm change from 1.76 to 1.72 and 1.68 with fixed concentration of heme, the absorbance at λmax=397 nm change from 2.74 to 2.63 and 2.55 with fixed concentration of Fc(Cys), and the absorbance at λmax=397 nm change from 1.82 to 1.58 and 1.49 with fixed concentration of heme, respectively.