In the clinic, numeration of circulating tumor cells (CTCs) plays a critical role in cancer diagnosis and treatment, but conventional CTC identification and counting that rely on specific antibodies to characterize a cell's surface antigens are costive and with limitations. Importantly, false positive or negative results may occur due to the high heterogeneity and epithelial-mesenchymal transition (EMT) of CTCs. Herein we demonstrate a novel and effective CTC detecting nanoprobe that could rapidly respond to the high level of endogenous H2O2 of CTCs and report the signal through fluorescence emission. Briefly, a hydrophobic coumarin-benzene boronic acid pinacol ester (Cou-Bpin) was grafted onto hydrophilic glycol chitosan (GC) to form an amphiphilic molecule, which further assembled into micellar nanoparticles in aqueous solution. This new nanoprobe was highly sensitive to H2O2 with a detection limit of 0.1 μM and could rapidly enter the cells within 30 min. Upon exposure to intracellular H2O2, the nanoprobe exhibited remarkable one-photon and two-photon luminescent characteristics, which were suitable for imaging of endogenous H2O2 of various human colorectal cancer cells and assist the identification of CTCs. Compared to a conventional CTC counting assay, the nanoprobe-based CTC numeration could overcome the false-negative findings due to the low expression of cytokeratin 19 (CK19). In a clinic test, CTC counting results based on the new nanoprobe match better to the postoperative pathological results of four clinic patients who had colorectal cancer at different stages.