We developed trimethyl lock quinone‐functionalized coumarin (probe 1) as a small fluorescent probe for the imaging of NQO1 activity in the live cells. NQO1 is highly expressed in most cancer cells and it is considered as a potential biomarker of NQO1‐related cancer. Probe 1 showed the high selectivity and rapid fluorescence response to the NQO1 activity. In addition, the probe is biocompatible and easily penetrates living cells. It was found that probe 1 provided relatively strong fluorescence in NQO1‐positive A549 cells, unlike NQO1‐negative NIH‐3T3 and MDA‐MB‐231 cells. Therefore, we demonstrated that probe 1 can detect NQO1 activity in real time in living cells and it can also distinguish different levels of NQO1 activity in various pathological cells.