Accurate measurements of linewidth standards, sidewalls and non-spherical nanoparticles performed at the nanoscale by means of Atomic Force Microscopy suffer from errors due to the tip shape and size. In order to reduce the uncertainty, the study here presented aims at investigating a bio-plant nanostructure, namely the Tobacco Mosaic Virus, as candidate reference tip characterizer.The TMV has a rod-shaped structure with a diameter of about 18 nm early reported from X-ray fibre diffraction, thus representing a reference at the nanoscale. When imaged by the AFM, the diameter of the TMV is determined as the top height of the rod from the reconstructed cross-section profile of isolated virions, deposited on a flat substrate like mica. A mean diameter of 16.5 nm, smaller than the nominal value by fibre diffraction measurements, is determined with our metrological AFM. Meanwhile, tip-samplesubstrate interactions are discussed with reference to experimental data and models in literature, in order to determine deformations and associated uncertainty of corrections, with which the difference between the AFM-reconstructed top-height diameter and the nominal value reduces to about 0.3 nm.Once the virus is fully characterised, a tip profile is estimated by the AFM-reconstructed cross-section profiles of the TMV. The approach used is to evaluate the tip-related enlargement from the nominal circle size, assumed undeformed, of the TMV cross-section profile. A good repeatability of the reconstructed tip shape is achieved from subsequent imaging of virions, while tip degradations are somewhat visible over the working time.