Mitochondria-associated ER membrane (MAM) is a structure where these calcium-regulating organelles form close physical contact sites for efficient Ca2+crosstalk. Despite the central importance of MAM Ca2+dynamics in diverse biological processes, directly and specifically measuring Ca2+concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca2+indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca2+-responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca2+indicator and quantitative structural marker specific for MAM. As a ratiometric Ca2+indicator, MAM-Calflux estimates steady-state MAM Ca2+levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca2+and the elucidation of abnormally accumulated MAM Ca2+from the neurons of Parkinson’s disease mouse model in both steady-state and stimulated conditions. Therefore, we propose that MAM-Calflux can be a versatile tool for ratiometrically measuring dynamic inter-organellar Ca2+communication.