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The previous activatable probe toward one analyte can still produce imprecise signal as a result of the challenge cased by accurate identification in complex biological environment. As smarter molecules, dual-locked probes are able to respond to two different analyte with one or more output signals, which display significantly reduced background interference and increased spatial resolution. In this study, a smart cysteine/lipid droplets sequence-activated dual-locked fluorescent probe CN–NO 2 has been designed and synthesized for the detection of tumor tissues. The recognition group of CN–NO 2 removed after reacting with cysteine (Cys) and undergoing intramolecular rearrangement to generate complete fluorophores. This structure had a strong solvent effect; it could recognize lipid droplets (LDs) in cells, thus exhibiting fluorescence without secondary molecular adjustment. The fluorescence of CN–NO 2 was enhanced 1752-fold after double unlocking. Importantly, the unlocking process only takes 10 min. Furthermore, the fluorescence of CN–NO 2 was amplified remarkable and anchored at LDs after reacting with Cys in A549 cells. Therefore, CN–NO 2 clearly indicated the site of mouse xenograft tumor in vivo. Such sequence-activated dual-locked fluorescent probes are envisioned to execute more analysis and diagnostic tasks giving these unique characteristics. • The CN–NO 2 was enhanced 1752-fold after cysteine/lipid droplets double unlocking, and the unlocking process only takes 10 min. [ABSTRACT FROM AUTHOR]