Human NAD(P)H: Quinone oxidoreductase 1 (NQO1) is over-expressed in many cancer cells. Thus, it is significant to establish a selective and sensitive method with high signal-to-background ratio for NQO1 detection and cancer diagnosis. Herein, an activatable biosensor based on water-soluble conjugated oligomer (OPFV-TLQ) was designed for highly sensitive detection of NQO1 and visualization imaging of cancer cells. Oligo (ethylene glycol) and the trimethyl lock quinone propionic acid group were modified on the side chain of OPFV-TLQ, which makes the probe possess good water-solubility and low fluorescence background signal, respectively. Interestingly, the benzoquinone can be reduced to hydroquinone by NQO1, which dramatically activates the fluorescence of OPFV-TLQ. Importantly, about 141-fold enhancement in intensity was obtained, which provides one of the highest signal-to-background ratio and the lowest detection limit of 0.068 ng/mL. Additionally, the catalytic constant was 67.71 μM−1 s−1 that is significantly higher than those of other probes, indicating the excellent catalytic efficiency of NQO1. Molecular docking simulation certifies that the probe is an excellent substrate of NQO1. Furthermore, the probe displays high selectivity and biocompatibility, and also has high-fidelity fluorescent signals in NQO1-positive cells. Thus, the biosensor holds great promise in biological applications of cancer cells/tissues imaging and cancer differentiation.