Although atomic force microscopy (AFM) technique has been employed to investigate the surface of biological samples, the interior regions are generally not accessible for imaging or probing. In this study we report a novel approach combining focused ion beam (FIB) and AFM to expose and study the interior cross section of biological samples [1] with an immediate application of studying rat whisker, a high-acuity tactile sensor. A rat whisker has been assumed to be homogeneous in material properties, as conventional mechanical measurements were restricted from accessing and measuring the interiors. In our experiment, prepared rat whisker samples were first milled with FIB milling orthogonal to the axis, and an installed lift-out shuttle microgripper was used to lift-out, manipulate and transfer the “cut-off” disc shape whisker sample, aiming for exposing the interior cross section of whisker followed by AFM probing. Adhesion and surface deformation for the cortex-medulla, cortex and cuticle regions of the whisker interior cross section were acquired, and site specific Young’s modulus values were calculated. A non-uniform modulus distribution inside the rat whisker was revealed, and finite element simulations based on the acquired data suggest that this pattern is a contributing factor to the high sensitivity of this tactile sensor [1]. The FIB lift-out protocol as well as characterisation methodologies developed, although only applied to the rat whisker in this study, can also be extended to a wide spectrum of biomechanical systems in future studies.