AbstractAMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But how AMPK activity is regulated at different subcellular locations for precise spatiotemporal control over metabolism is unclear. Here we present a sensitive, single-fluorophore AMPK activity reporter (ExRai AMPKAR), which reveals distinct kinetic profiles of AMPK activity at the mitochondria, lysosome, and cytoplasm. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) results in slower AMPK activity at lysosomes but does not affect the response amplitude at lysosomes or mitochondria, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further identify a mechanism for AMPK activity in the nucleus, which results from cytoplasmic to nuclear shuttling of AMPK. Thus, ExRai AMPKAR enables illumination of the complex subcellular regulation of AMPK signaling.