In this research, the interaction of [CuL(DMF)], [NiL(DMF)] and [VOL(DMF)] (where L = ((E)-4-((2-amino-5-nitrophenylimino)methyl)benzene-1,3-diol)) complexes derived from tridentate Schiff base ligand with bovine serum albumin (BSA) and DNA was investigated via electronic absorption and fluorescence spectroscopy. The Ultraviolet-Visible (UV-Vis) spectra exhibited an isosbestic point for the complexes through titration with DNA. The experimental results showed the presence of intercalation interaction between the complexes and calf-thymus DNA (CT-DNA). The interaction of BSA protein and complexes was significant. The recorded florescence spectra of complexes interacting with DNA and BSA revealed the static quenching manner. The free binding energies of complexes and their interaction modes with DNA and BSA were determined by the molecular docking. MTT-dye reduction technique was applied to define cytotoxicity of [NiL(DMF)], [CuL(DMF)] and [VOL(DMF)] complexes against breast cancer 4T1 and colon carcinoma C26 cell lines. The [VOL(DMF)] complex had cytotoxic activity against 4T1 and C26 cell lines.