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Effects of substrate hydrophobicity/hydrophilicity on height measurement of individual ds-DNA molecules are investigated with tapping mode atomic force microscopy (TMAFM) and vibrating mode scanning polarization force microscopy (VSPFM). The measured heights of ds-DNA on hydrophobic highly oriented pyrolytic graphite (HOPG) are remarkably less than those on hydrophilic bare mica and Ni2+ treated mica in both TMAFM and VSPFM. By analysing the results, we propose that the hydrophobicity/hydrophilicity of substrate can greatly influence the height measurement of DNA molecules.