The new alkoxime ligand (E)-anthracene-9-carbaldehyde O-methyl oxime ( 1 ) derivative of 9-anthraldehyde and its palladium(II) complexes trans -Pd (C 15 H 10 NOCH 3 ) 2 (OAc) 2 ( 2 ) and [ cis -Pd (C 15 H 10 NOCH 3 )(Py) 2 (OAc)](OAc) ( 3 ) were synthesized and characterized by spectroscopic techniques including FT-IR, NMR and elemental analysis. The structure of alkoxime ligand characterized by X-ray crystallography. Then interaction of them with calf thymus (CT) DNA and bovine serum albumin (BSA) was explored using absorption and emission spectral methods. Ligand ( 1 ) and complex ( 3 ) bind strongly to DNA via intercalation and complex ( 2 ) bind to it by groove binding mode. The binding modes were further confirmed by CD spectra. BSA binding aspects of synthesized compounds have been studied through various biophysical techniques. Furthermore, they were assessed for in vitro cytotoxicity testing on the CT-26 and MDA-MB231cell lines was carried out. The three compounds displayed better cytotoxic profiles against CT-26 than MDA-MB231 cancer cell lines.