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Since adenosine triphosphate (ATP) and reactive oxygen species (ROS) are closely related to neuron functions and the pathogenesis of Alzheimer's disease, herein, the first example of using a dual-responsive zeolitic imidazolate framework-90 (ZIF-90) to fabricate a single fluorescent probe, CuNCs/ZIF@P1-P2/ZIF, was reported, which specifically and sensitively responded to intracellular level fluctuations of ATP and ROS both in neuronal and tumor cells without obvious cross-talking. Through a stepwise in situ growth method, ROS-responsive glutathione-stabilized copper nanoclusters (GSH-CuNCs) and double tagged ATP aptamer hybrid chain (P1-P2) were prepared and respectively encapsulated into the inner and outer ZIF-90. The obtained CuNCs/ZIF@P1-P2/ZIF probe as a tandem system could firstly respond to ATP in an "off-to-on" mode, in which, the quenched fluorescence of FAM by BHQ1 attached on P2 was recovered through the competitive displacement between ATP and ZIF-90, leading to the release of FAM from ZIF-90. Upon collapse of the outer ZIF-90, the strong emission of CuNCs enclosed in the inner ZIF-90 was subsequently quenched in the presence of ROS. Accompanied with high sensitivity, selectivity and accuracy, the resultant fluorescent probe exhibited good performance and properties for tracking the level variations of ATP and ROS in SH-SY5Y and Hela cells under different stimulations. This strategy highlighted the potential of the ZIF-90 based nanocomposite for multi-targets detection, and also provided a value tool for the early diagnosis of diseases. • A dual-responsive ZIF-90 based nanocomposite was prepared. • An effective strategy for ATP and ROS combined detection was developed. • The composite selectively responded to ATP and ROS without cross-talking. • Imaging in living SH-SY5Y cells under different stimulations was realized. [ABSTRACT FROM AUTHOR]