Thiophenol and its derivatives pose a significant threat to the environment and biological systems due to their toxic nature. The imperative need for their detection prompted the development of a "turn-on" fluorescent probe, NI-TPA-1, incorporating 1,8-naphthalimide-triphenylamine as the emission groups and 2,4-dinitrobenzene as the reactive site. Upon introducing 4-methoxythiophenol (MTP), the fluorescence intensity of NI-TPA-1 exhibited a remarkable 18-fold increase, achieving detection limits as low as 20 nM in aqueous solutions. The photo-induced electron transfer (PET) response mechanism was elucidated through density functional theory (DFT) calculations, providing valuable insights into the sensing mechanism of the probe. Furthermore, the practical applicability of NI-TPA-1 was demonstrated through its successful detection of thiophenol in real water samples. The versatility of the probe was further showcased in living cells imaging, highlighting its potential for in vivo applications. This research not only presents a robust solution for the sensitive and selective detection of thiophenol but also underscores the broader significance of NI-TPA-1 in environmental and biomedical contexts.