Cytochrome P450 1B1 (CYP1B1) was found to be universally expressed in various tumors. Herein, we reported near-infrared fluorescent imaging probes for tumor detection via visualizing CYP1B1. After introducing the linker to a CYP1B1 selective inhibitor we found previously, we got the resulting compound 5b which kept strong inhibition ability against CYP1B1 (IC50 = 8.7 ± 1.2 nM) and high selectivity. Then, in vitro microscope studies and cell binding assay of probes indicated that the corresponding probe 6b could specifically be accumulated in CYP1B1 overexpressed colorectal cancer cell HCT-15 and showed satisfying binding affinity to target. During the in vivo noninvasive optical imaging, 6b was proved to rapidly lighten tumor in vivo as early as 6 h after injection. This work is the first attempt to visualize CYP1B1 for noninvasive imaging of tumor which could provide new approach for tumor diagnosis.