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The abnormal concentration of adenosine 5-triphosphate (ATP) in cancer cells is closely related to the process of tumorigenesis and development. Therefore, rapid and accurate detection of ATP level in and out of cells is of great significance. Graphene oxide (GO) is widely used owing to its advantages of low toxicity, large specific surface area, easy functionalization, efficient and stable loading of DNA nanoprobes into cells. However, ssDNA bound to GO by physical adsorption is easily displaced by biomolecules in complex biological environment, leading to false positive signals. Based on this, a novel GO-DNA nanoprobe is proposed and applied to the detection of ATP and targeted delivery of the anticancer drug doxorubicin hydrochloride (DOX). Double stranded DNA (dsDNA) is formed by hybridization of aptamer of ATP with complementary strand, and then DOX is loaded by G-C base pair to form dsDNA-DOX. dsDNA-DOX is adsorbed on GO surface through polyA sequence extended by complementary strand to construct GO-dsDNADOX nanoprobes, which could greatly reduce false positive signal caused by the physical adsorption of biomolecules in complex environment. The specific binding of ATP to aptamer can lead to the release of DOX, and quantitative analysis of ATP is achieved according to its fluorescence “off-on”. DOX fluorescence intensity shows a good linear relationship with the ATP content within the range of 0. 08~8. 0 mmol/L, and the linear equation is IF=3. 0897c+129. 08. The detection limit (3σ/k) is 0. 059 mmol/L, and the method has good selectivity and anti-interference ability. Cytotoxicity and fluorescence imaging results show that the DOXloaded nanoprobes have significant drug release in MCF-7 cells and significantly induce apoptosis. This study establishes a label-free, simple and rapid method for ATP content detection and realizes targeted drug delivery by the high content of ATP in cancer cells, which reduces the toxic and side effects on normal cells and provides a new idea for cancer treatment. [ABSTRACT FROM AUTHOR]