Human cytochrome P450 1A1 (CYP1A1) enzyme is involved in the oxidation of various procarcinogenes into active carcinogenic metabolites in the phase I of biotransformation and initiates the carcinogenic process. Here, a novel ultrasensitive CYP1A1-specific fluorescent probe (DPCl) was investigated deeply which has O-chloroethyl group as a trigger for CYP1A1 activity and a dicyanoisophorone derivative fluorophore with a 673 nm emission. The size of the O-chloroethyl group is suitable for the cavity volume of the active sites of CYP1A1 and the lipophilicity and polarity of the group make the probe suitable for oriented at the actives sites. Hence, this near-infrared fluorescent probe reveals ultrasensitive sensitivity (minimum detectable limit is 0.026 nM), excellent specificity, and low toxicity in the process of target enzyme monitoring under simulated physiological condition, living cells and zebrafish. The success of this strategy is rely on intermediate product (DPOH) after dechloroethylation of DPCl is NIR-illuminant platform with a prominent Stokes Shift (118 nm), which is the first report of a NIR fluorescent probe for detecting CYP1A1. Anyhow, this probe provides support for clinical studies of CYP1A1 cell screening inhibitors and real-time tracking of enzyme activity in vivo.