In the present study, we reported the syntheses and investigation of the modes of binding to DNA and serum albumins of the two ethidium derivatives containing ethyl and methyl chloroformate of both amines at 3-and 8- positions. The interactions of calf thymus DNA (ct-DNA) and serum albumins (BSA and HSA) with the two derivatives, E4 and E5, were investigated using fluorescence quenching method and UV-visible absorption spectra. The obtained data suggest that used derivatives can interact strongly with DNA and serum albumins; there are the formation of derivative-DNA and serum albumin complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern-Volmer quenching constants, binding constants, binding sites and the corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The results of absorption, ionic strength and iodide ion quenching suggested that the interaction mode of E4 and E5 with DNA was groove binding.