(4) (glyha 2− = dianion glycinehydroxamic acid, alaha 2− = dianion alaninehydroxamic acid, bpy = 2,2 -bipyridine, phen = 1,10-phenanthroline) have been successfully synthesized and characterized by X-ray single crystal diffraction. The interactions of these complexes with calf thymus DNA (CT-DNA) were studied through UV spectroscopy, fluorescence spectroscopy, and circular dichroism. The results revealed that complexes 1-4 could interact with CT-DNA through intercalation. Interactions of all complexes with bovine serum albumin (BSA) were confirmed by the docking study to quench the intrinsic fluorescence of BSA in a static quenching process. Furthermore, the in vitro cytotoxic effect of the complexes was also examined on four tumor cell lines, including human lung carcinoma cell line (A549), human colon carcinoma cell line (HCT-116), human promyelocytic leukemia cell (HL-60) and cervical cancer cell line (HeLa). All complexes exhibited different antitumor activities.