Copper-based bis(hydroxy naphthaldehyde) complex with axial mono aqua-coordination was synthesized and thoroughly characterized by various spectroscopic{IR, UV-vis, EPR}, ESI-Mass and single X-ray crystallographic studies. The single X-ray crystallography of the complex revealed the square pyramidal coordination geometry with P21 space group with axial water molecule ligated to copper centre. The geometry of the complex was further validated by DFT calculations, which was in accordance with other spectroscopic studies. The binding profile of the complex with ct-DNA and tRNA was carried out by employing various biophysical (absorption, fluorescence, circular dichroism, morphological studies) and computational studies (DFT, molecular docking). The experimental results revealed efficient binding of the complex with both ct-DNA and tRNA primarily, via non-covalent interactions. The binding studies K(b) and K values revealed 10-fold greater binding affinity of the complex for tRNA as compared to ct-DNA. The in silico molecular docking further validated the interaction of complex within the hydrophobic pocket of ct-DNA and tRNA. The concentration and time dependent cleavage studies of DNA and tRNA were performed by employing gel electrophoresis assay. The cytotoxic activity of the complex was performed on a panel of human cell lines viz., leukemia (K-562), pancreatic (MIA-PA-CA-2), hepatoma (Hep-G2), cervical (HeLa), and breast (MDA-MB-231) by SRB assay. The complex exhibited selectively remarkably good cytotoxic potential on leukemia (K-562), cervical (HeLa) and hepatoma (Hep-G2) cancer cell lines.Communicated by Ramaswamy H. Sarma.