Cysteine is a significant biothiol and plays a crucial role in physiological and clinical diagnosis. In the current research, we report a novel near-infrared (NIR) fluorescence sensor derived from triphenylamine-isophorone conjugate for the detection of cysteine. In the sensing pathway, an NIR fluorescence emitting moiety is released after a fast addition-cyclization of cysteine to the methacrylate group, which acts as an emission quencher. The detection platform demonstrates an excellent linear response to cysteine. It has been determined that the probe displays a fast response time (about 4 min), a low detection limit (12.4 nM), and a high signal-to-noise ratio. Easy and quick practical applications such as visual thin layer chromatography (TLC) plate and cotton swab have been established. Also, the current promising approach is effectively applied for the bio-imaging of cysteine in yeast cells and for drug analysis. Thus, this strategy allows the current probe to sense selectively cysteine over other biothiols in 'turn-on' mode.