Abstract G‐quadruplex (G4) structures are significant therapeutic targets for diseases such as cancer, but developing fluorescent probes capable of targeting mitochondrial and nuclear parallel G4 DNA while achieving precise imaging and therapeutic functions remains challenging. Herein, a series of pyridinium cationic fluorescent probes with varying alkyl chain lengths is synthesized for organelle‐specific targeting. Among them, probe PCP initially accumulated in mitochondria, where its interaction with mitochondrial G4 DNA disrupted the cell cycle and induced reactive oxygen species imbalance, ultimately causing mitochondrial damage. Subsequently, PCP migrated from mitochondria to the nucleus, where its enhanced fluorescence response is activated by increased viscosity from cellular damage, enabling in situ self‐reporting of apoptosis. After being encapsulated within homotypic cell membranes to form PCP‐ML NPs, the resulting nanoprobes demonstrated efficient targeting toward MCF‐7 cells. Owing to this specific targeting, at a concentration of 10 µ m , the PCP‐ML NPs induced apoptosis in 72.37% of the cells and significantly inhibited their proliferation. This work presents a useful strategy for applying parallel G4 DNA‐targeting fluorescent probes in cancer imaging and therapy.