<p>Precisely elucidating the apoptotic signaling pathway is crucial for monitoring apoptosis, advancing drug discovery, and improving treatments for human diseases. However, it is challenging to directly describe these pathways, due to the fact that individual signaling molecule detection cannot establish a causal relationship. In this paper, we developed a dual responsive DNA nanomachine capable of simultaneously detecting both upstream and downstream signaling molecules in an apoptotic signaling pathway. The walking machine is constructed by anchoring a few blocker-myc/blocker-Cyt c/DNAzyme triple DNA strands containing complementary domains for c-Myc mRNA (blocker-myc) and an aptamer for Cyt c (blocker-Cyt c), together with an abundance of Cy5-labeled DNA-RNA chimeric substrates onto gold nanoparticles (AuNPs) through an Au–S bond. Upon activation by the c-Myc mRNA and Cyt c, the “AND” logic gate designed by blocker-myc/blocker-Cyt c/DNAzyme triple DNA strands could be “open,” resulting in the autonomous movement of the DNAzyme along the AuNP and outputting a fluorescence signal. Unlike conventional methods that detect single molecules, this dual responsive DNA walking nanomachine achieves direct profiling of a specific signaling pathway by correlating two key signaling molecules. Our strategy holds significant potential for investigating apoptotic mechanisms, elucidating signaling pathways, and facilitating drug screening.</p>