A mechanism-based spectrophotometric titration of nano-alumina powder with calf thymus ds-DNA was carried out at two temperature levels with a view to generating possible mode of interaction between this industrially useful nanopowder and DNA. Varying molar ratio combinations of DNA with the nano-alumina powder were studied at 37 °C and 45 °C after 30 min incubation period. The UV–Vis spectral data were acquired from 200 to 650 nm. The effect of the nanopowder on the most prominent wavelength maximum of DNA at 280 nm was studied. Stoichiometric ratio determination was determined using Job’s method of continuous variation. The formation constants between the ds-DNA and alumina nanopowder were calculated from a modified Benesi–Hildebrand equation for complex formation. In order to understand the spectral changes observed, thermodynamic parameters were evaluated such as the Gibbs free energy, enthalpy and entropy changes. The alumina nanopowder produced a pronounced hypochromic effects on the spectra of ds-DNA at all mole ratios relative to 100% DNA. There was enhanced effect at the elevated temperature of 45 °C with slight hypsochromic shift. These suggest some level of interaction with ds-DNA. The plot of absorbance of complexes at the respective λmax produced two points of inflection which suggest that the two atoms of aluminum in Al2O3 were interacting with DNA molecule. The formation constant varied directly with increase in temperature exhibiting an endothermic reaction. Evaluation of the thermodynamic parameters showed a large free energy change with positive enthalpy and small positive entropy. This confirms a dative covalent bonding interaction. The simple method presented in this research can be used to screen suspected genotoxins especially following occupational exposures.