AbstractTraditionally, nanomaterial profiling using a single-molecule-terminated scanning probe is performed at the vacuum–solid interface often at a few Kelvin, but is not a notion immediately associated with liquid–solid interface at room temperature. Here, using a scanning tunnelling probe functionalized with a single C₆₀ molecule stabilized in a high-density liquid, we resolve low-dimensional surface defects, atomic interfaces and capture Ångstrom-level bond-length variations in single-layer graphene and MoS₂. Atom-by-atom controllable imaging contrast is demonstrated at room temperature and the electronic structure of the C₆₀–metal probe complex within the encompassing liquid molecules is clarified using density functional theory. Our findings demonstrates that operating a robust single-molecular probe is not restricted to ultra-high vacuum and cryogenic settings. Hence the scope of high-precision analytics can be extended towards resolving sub-molecular features of organic elements and gauging ambient compatibility of emerging layered materials with atomic-scale sensitivity under experimentally less stringent conditions.