Protein phosphatase 2A (PP2A), a pivotal serine/threonine phosphatase, plays a crucial role in cellular regulation and tumor suppression. Dysregulation of PP2A complex, particularly the Aα subunit and B56 family, is linked to malignancies through altered substrate interactions, exemplified by c-MYC dynamics. Given the challenges in identifying PP2A substrates—owing to the enzyme’s expansive substrate range, transient interaction profiles, and complex regulatory mechanisms—we employed bioluminescence resonance energy transfer (BRET) sensors. These advanced molecular tools facilitate the real-time detection of protein-protein interactions within live cells. This investigation details the creation and application of a novel PPP2R5A (B56α) BRET sensor tailored for cytosolic and nuclear environments, effectively distinguishing specific PP2A interactions. The nuclear sensor, enhanced with a nuclear localization signal, enabled probing of targets like c-MYC. The dual compartmental utility of these sensors underscores their significant potential in elucidating PP2A’s regulatory roles and their implications in oncogenesis. Our study highlights the efficacy of BRET sensors in formulating precision therapeutic strategies. This advancement provides a robust framework for deeper investigations into the multifaceted roles of PP2A in both normal physiological and pathological contexts, paving the way for future explorations into its intricate molecular interactions.