High-purity heavy water (D 2 O) is a strategic material owing to its important application in the fields of nuclear energy and scientific research. D 2 O always tends to get contaminated by H 2 O owing to its strong hygroscopicity. Herein, a bimetallic hexanuclear Ln(III) cluster-based metal-organic framework ( Eu 0.5 Tb 0.5 -TZB-MOF ) has been synthesized for fluorescence sensing of the D 2 O-H 2 O binary mixtures. Eu 0.5 Tb 0.5 -TZB-MOF can be used to immediately differentiate D 2 O or H 2 O via fluorescent color responses that are obvious to the naked eye and allow for quantitative ratiometric analysis using simple spectrophotometry. Fluorescence titration experiments demonstrate that both trace H 2 O in D 2 O and trace D 2 O in H 2 O can be quantitatively detected. Mechanistic studies demonstrate that the weaker vibrational quenching of the O-D oscillator compared to the O-H oscillator, in addition to the terbium-to-europium energy transfer, triggered the fluorescence signal response.