Objective To investigate the in situ visualization imaging and the in vivo distribution of the nearinfrared fluorescent quantum dots(QDs) epidermal growth factor receptor(EGFR) monoclonal antibody(mAb) probe in head and neck squamous cell carcinoma(HNSCC). Methods QDs with an emission wavelength of 800 nm(QD800) was conjugated with EGFR mAb to produce QD800-EGFR mAb. QD800-EGFR mAb was co-cultured with BcaCD885 squamous cancer cell line for 30 min and observed with laser scanning confocal microscope(LSCM). QD800-EGFR mAb was injected into HNSCC animal model through the tail vein, and the in situ visualization imaging and the in vivo distribution of QD800-EGFR mAb was analyzed at different time points. Results BcaCD885 squamous cell carcinoma in the head and neck can be imaged clearly and visually after intravenous injection of QD800-EGFR mAb probe, these fluorescence signals lasted for 24 h. The most complete tumor images with maximal signal-to-noise ratio were observed from 30 min to 6 h after injection of the probe. In vivo tissue distribution studies demonstrated that QD800 aggregated mostly in liver. QD800 aggregation decreased with time in tumors, and QD800 didn’t aggregate in heart, brain, intestine, lung and stomach. Conclusion The QD800-EGFR mAb probe can clearly produce visual images in head and neck cancer. It has promising prospects in visualization in vivo imaging and individual treatment of head and neck cancer.