Detection of highly toxic thiophenols in biological or environmental systems is of great importance. Therefore, fast, reliable, and sensitive probes are needed to detect thiophenols. Herein, a novel triphenylamine conjugated dicyanoisophorone-based near infrared fluorescence probe is reported to determine trace thiophenol (PhSH) levels. The probe demonstrates a distinct “turn-on” fluorescence response to thiophenol among the tested analytes and its quantum yield (Φ) increases from 0.011 to 0.142. It has low cytotoxicity with cell viability of 90–100% up to 10.0 μM of the probe, a strong anti-interference capability, a large Stokes shift (150 nm), and a fast response time (<1 min). In addition, the probe exhibits a good linear response to PhSH over the range from 0 to 15.0 μM with a detection limit of 32.3 nM (R2 = 0.9978). The detection process is also confirmed through HPLC. The practical applicability of the probe is proved by a smartphone platform, TLC kit, plant tissue imaging, soil assay, tap, and lake water analysis with good recovery values (92.3–117%), and concentration-dependent live cell bioimaging PhSH from 5.0 to 15.0 μM. Therefore, the present probe is a robust candidate for monitoring PhSH levels in biological and environmental systems.