Abstract Despite their growing popularity, many fundamental properties and applications of MXene materials remain underexplored. Here, the nonradiative energy transfer properties of 2D titanium carbide MXene are investigated and their application in single‐molecule biosensing is explored for the first time. DNA origami positioners are used for single dye placement immobilized by a specific chemistry based on glycine‐MXene interactions, allowing precise control of their orientation on the surface. Each DNA origami structure carries a single dye molecule at predetermined heights. Single‐molecule fluorescence confocal microscopy reveals that energy transfer of an organic emitter (ATTO 542) on transparent thin films made of spincast Ti 3 C 2 T x flakes follows a cubic distance dependence, where 50% of energy transfer efficiency is reached at 2.7 nm ( d 0 ). MXenes are applied as short‐distance spectroscopic nanorulers, determining z distances of dye‐labeled supported lipid bilayers fused on MXene's hydrophilic surface. Hydration layer (2.1 nm) and lipid bilayer thickness (4.5 nm) values that agree with the literature are obtained. These results highlight titanium carbide MXenes as promising substrates for single‐molecule biosensing of ultrathin assemblies, owing to their sensitivity near the interface, a distance regime that is typically inaccessible to other energy transfer tools.