The development of highly sensitive and precise imaging techniques capable of visualizing crucial molecules at the subcellular level is essential for elucidating mitochondrial functions and uncovering novel mechanisms in biological processes. However, traditional molecular imaging strategies are still limited by off-mitochondria signal leakage because of the "always-active" sensing mode. To address this limitation, we have developed a light-triggered activation sequence activated plasmonic DNAzyme walker (PDW) for accurate subcellular molecular imaging by the combination of an organelle localized strategy, upconversion nanotechnology, and a plasmon enhanced fluorescence (PEF) technique. Exploiting the advantage of light activation enables precise control over when and where to activate the probe's sensing function, effectively reducing off-mitochondria signal leakage as validated by the dynamic monitoring of changes in off-mitochondria signals during the mitochondrial entry process. Furthermore, by leveraging the PEF capability of triangular gold nanoprisms (Au NPRs), the fluorescence intensity can be enhanced by approximately 11.9 times, ensuring highly sensitive and accurate subcellular molecular imaging.