Background: Oral cancer screening is important for early detection and early treatment, which help improve survival rates. Biopsy is the gold standard for a definitive diagnosis but is invasive and painful, while fluorescence visualization is non-invasive, convenient, and real-time, and examinations can be repeated using optical instruments. The purpose of this study was to clarify the usefulness of fluorescence visualization in oral cancer screening. Methods: A total of 502 patients, who were examined using fluorescence visualization with optical instruments in our hospitals between 2014 and 2019, were enrolled in this study. The final diagnosis was performed by pathological examination. Fluorescence visualization was analyzed using subjective and objective evaluations. Results: Subjective evaluations for detecting oral cancer offered 96.8% sensitivity and 48.4% specificity. Regarding the objective evaluations, sensitivity and specificity were 43.7% and 84.6% for mean green value, 55.2% and 67.0% for median green value, 82.0% and 44.2% for coefficient of variation of value, 59.6% and 45.3% for skewness, and 85.1% and 75.8% for value ratio. For the sub-analysis of oral cancer, all factors on objective and subjective evaluation showed no significant difference. Conclusions: Fluorescence visualization with subjective and objective evaluation is useful for oral cancer screening.