Cytochrome P4501A1 (CYP1A1), a heme-containing monooxygenase, is of particular importance for human health because of its vital roles in the metabolic activation of pro-carcinogenic compounds to the carcinogens. Deciphering the relevance of CYP1A1 to human diseases and screening of CYP1A1 modulators require reliable tool(s) for probing this key enzyme in complex biological matrices. Herein, a practical and ultrasensitive fluorescence-based assay for real-time sensing CYP1A1 activities in biological systems has been developed, via designing an isoform-specific fluorogenic sensor for CYP1A1 (CHPO). The newly developed fluorogenic substrate for CYP1A1 has been carefully investigated in terms of specificity, sensitivity, precision, quantitative linear range and the anti-interference ability. The excellent selectivity, strong anti-interference ability and fast response kinetics, making the practicability of CHPO-based CYP1A1 activity assay is better than that of most reported CYP1A1 activity assays. Furthermore, CHPO has been successfully used for imaging CYP1A1 activities in living cells and human tissues, as well as for high-throughput screening of CYP1A1 inhibitors using tissue preparations as enzyme sources. Collectively, this study provided a practical fluorogenic sensor for real-time sensing CYP1A1 in complex biological systems, which would strongly facilitate the investigations on the relevance of CYP1A1 to human diseases and promote high-throughput screening of CYP1A1 modulators for biomedical applications.