We measured and quantified the local electric polarization properties of ultrathin (∼5 nm) biolayers on mm-thick mica substrates. We achieved it by scanning a sharp conductive tip (<10 nm radius) of an electrostatic force microscope over the biolayers and quantifying sub-picoNewton electric polarization forces with a sharp-tip model implemented using finite-element numerical calculations. We obtained relative dielectric constants εr = 3.3, 2.4 and 1.9 for bacteriorhodopsin, dioleoylphosphatidylcholine (DOPC) and cholesterol layers, chosen as representative of the main cell membrane components, with an error below 10% and a spatial resolution down to ∼50 nm. The ability of using insulating substrates common in biophysics research, such as mica or glass, instead of metallic substrates, offers both a general platform to determine the dielectric properties of biolayers and a wider compatibility with other characterization techniques, such as optical microscopy. This opens up new possibilities for biolayer research at the nanoscale, including nanoscale label-free composition mapping.