The amount of protein thiols play a crucial role in maintaining the cellular redox homeostasis and have significant implications to indicate a series of diseases. Therefore, it is necessary to develop an ideal probe for protein thiol detection in a simple and readily implementable method. Consequently, a water-soluble and incubate-free fluorescent environment-sensitive probe DMTs-OCC was synthesized using 7-diethylamincoumarin as the fluorophore and 4-(5-Methanesulfonyl- [1,2,3,4]tetrazol-1-yl)-phenol (MSTP) as a thiol receptor reagent. The blue-shift emission spectra of probe DMTs-OCC was observed by ultrafast binding to protein sulfhydryl groups from the excited intramolecular charge transfer (ICT) to the twisted intramolecular charge transfer (TICT) conversion process in aqueous solution. The experimental results showed that probe DMTs-OCC exhibited an excellent selectivity to protein thiols and biocompatibility in aqueous solution, as well as terrific cell membrane permeability which enabled the successful visualization of BSA protein thiol in living cells. Moreover, no excess probe was cleaned and no incubation time was needed in cell experiments. Therefore, it could provide a new method to the construction of fluorescent probes for protein thiols labelling and visualization.