An activatable mitochondria-targeted fluorescent probe Hcy-OPD was synthesized for the detection of methylglyoxal (MGO). For the introduction of a preorganized isopropylamino group on the aromatic o-diamine framework to regulate the hindrance effect, Hcy-OPD showed high selectivity and sensitivity (0.22 μM) for monitoring MGO. The probe can be applied successfully in the imaging of exogenous and endogenous MGO in living cells.