In novel synthetic 28 4-arylamino-6-fluoro quinazoline derivatives, compound 3a displayed the most remarkable inhibitory activities against tumor cells (IC<sub>50</sub> values ranging between 0.71 and 2.30 μM) in vitro. Importantly, 3a obviously inhibited the proliferation and metastasis of A549 cells in a zebrafish xenograft model, while also mediated cell apoptosis and G0/G1-phase cell cycle arrest in A549 cells. Interestingly, 3a had excellent fluorescence at 439 nm (λ<sub>ex</sub> = 375 nm) in DMSO and at 428 nm (λ<sub>ex</sub> = 372 nm) in 0.5% DMSO-phosphate buffer, and the self-fluorescent characteristic revealed 3a itself accumulates in the mitochondria of A549 cells, which suggested the antitumor process of 3a may involve the mitochondrial apoptotic pathway. The hypothesis was verified by the increase of the intracellular reactive oxygen species, the decrease of mitochondrial membrane potential, the release of cytochrome C from the mitochondria into the cytoplasm, and the cascade activation of caspase-9 and caspase-3 when A549 cells were treated with 3a. This work has great implications for further development of anticancer agents that can be enriched in mitochondria and can be tracked in real-time in biological systems due to the ideal fluorescence.