Among the physiological and pathological sulfur-containing species, cysteine (Cys) is the most typical one which is an important component of the REDOX system in vivo. Monitoring the level of Cys from other competing species seems quite important in pre-clinical diagnosis and therapeutic evaluation. Herein, we developed a selective fluorescent probe, BPCys, for Cys from the benzo[a]phenazin backbone which had the potential of anti-cancer potency. BPCys suggested advantages including high specificity (40 fold over other species), high sensitivity (detection limit: 18 nM), wide pH adaptability (6.0–11.0) and in particular, the anti-cancer effect. Biological assays and in silico simulation hinted the potency of the detecting product on Topoisomerase I/II. In brief, this study raised a practical strategy for monitoring the Cys level in living cells, especially in cancer models with its anti-cancer potential, thus opened the mind of exploring more specific tool for specific applications.