As a monolayered version of nanoscale metal-organic frameworks (nMOFs), nanoscale metal-organic layers (nMOLs) represent an emerging class of highly tunable two-dimensional materials for hierarchical functionalization and with facile access to analytes. Here we report the design of the first nMOL-based biosensor for ratiometric pH and oxygen sensing in mitochondria. Cationic Hf12-Ru nMOL was solvothermally synthesized by laterally connecting Hf12 secondary building units (SBUs) with oxygen-sensitive Ru(bpy)32+-derived DBB-Ru ligands (bpy = 2,2'-bipyridine). The Hf12-Ru nMOL was then covalently functionalized with pH-sensitive fluorescein isothiocyanate and pH/oxygen-independent Rhodamine-B isothiocyanate through thiourea linkages to afford Hf12-Ru-F/R as a mitochondria-targeted ratiometric sensor for pH and O2 in live cells. High-resolution confocal microscope imaging with Hf12-Ru-F/R revealed a positive correlation between pH and local O2 concentration in mitochondria. Our work shows the potential of nMOL-based ratiometric biosensors in sensing and imaging of biologically important analytes in live cells.