Although various fluorescent-based nanoparticles are treated as cellular imaging probes, approaching the construction of a biocompatible subcellular imaging probe is challenging. At the same time, the recognition of wasted pharmaceutical drugs by some fluorescent nanoprobes is important and urgently required. We report a “structural memory” concept for simple one-pot synthesis of bright green fluorescent (quantum yield of up to 61%) carbon dots (C-dots) from triphenylphosphonium (TPP) as a carbon precursor that will simultaneously act as an effective vehicle for mitochondria labeling in cancer cells and as a selective tetracycline sensor. The ubiquitous TPP residues upon the C-dots’ surface easily recognize the cellular mitochondria. Tetracycline has been selectively and instantaneously detected through rapid fluorescence on-off response from C-dots where other drugs remained silent in nature, even after longer incubation. This quenching response is ascribed to the static quenching effect and position of functional groups of the targeted drug which can play a dominating role. The reason for strong fluorescence exhibition from C-dots has been well explained by considering different factors. Such types of C-dots have been shown to be universal mitochondria-targeting nanoprobes, non-cytotoxic, and effective as a tetracycline detector. This finding should open a new avenue for in-vivo therapeutic application and sensing of pharmaceutical drugs in real clinical applications. © 2021 Wiley-VCH GmbH