Herein, a novel 1,8-naphthalimide-based fluorogenic compound, chemoprobe NASP, was designed and developed as a sensor platform for the selective detection of biologically important cysteine (Cys) over glutathione (GSH) and homocysteine (Hcy) in living-cells. In the presence of Hcy or Cys, the chemoprobe NASP demonstrated a remarkable response in EtOH : H₂O (90 : 10, v/v, 0.0670 M PBS buffer pH = 7.0) media at 416 nm (λ_max), while the competitive amino acids showed no significant emission. To assess the fluorescence parameters, a validation study was also carried out. The chemoprobe NASP offered a great sensitivity towards Cys and Hcy with ultra-low detection limits (Cys, 9.06 nM; Hcy, 57.5 nM), and it was highly selective over natural amino acids. The proposed sensing mechanism was confirmed using the MALDI-TOF-MS method. The molecular and electronic structures of the chemoprobe NASP and NASP-OH(Cys) were confirmed through DFT study. More importantly, the bio-imaging of intracellular Cys and Hcy by the chemoprobe NASP was successfully applied with low cytotoxicity in living cells (human normal hepatocytes (THLE2) and human hepatocellular carcinoma (HepG2) cells). The results indicated that the developed chemoprobe will be a promising fluorogenic sensor for the diagnosis of biothiol-related illnesses.