OBJECTIVE: ɤ-glutamyltranspeptidase is an enzyme expressed in various malignancies including lung cancer. It rapidly activates non-fluorescent ɤ-glutamyl hydroxymethyl rhodamine green to highly fluorescent hydroxymethyl rhodamine green. The resultant tumor fluorescence is therefore an indicator of cellular ɤ-glutamyltranspeptidase activity. We have explored the use of ɤ-glutamyl hydroxymethyl rhodamine green as an intraoperative imaging tool for visualizing cancers. Herein, we evaluated the potential of the tumor fluorescence as a postoperative prognostic indicator. METHODS: We included patients with non-small cell lung cancer who had undergone radical resection from 2012 to 2014 in the study. We assessed the fluorescence intensity of the resected tumor and normal lung tissue by ex vivo imaging using ɤ-glutamyl hydroxymethyl rhodamine green. RESULTS: Sixty-seven patients were eligible for the study (adenocarcinomas, n = 44; squamous cell carcinoma, n = 14; other histologies, n = 8). The pathological stages were I, II, III, and IV in 39, 15, 12, and 1 patient, respectively. Based on the fluorescence of the tumor tissue, the patients were divided into high fluorescence (n = 33) and low fluorescence (n = 34) groups. The 5-year overall survival rate was significantly higher in the high fluorescence group (72.7%) compared to the low fluorescence group (47.1%, P = 0.025). Similarly, pathological stage I patients of the high fluorescence group had higher 5-year overall survival (85.7% vs. 44.4%, P = 0.009) and recurrence-free survival (76.2% vs. 44.4% P = 0.044) rates compared to those of the low fluorescence group. CONCLUSIONS: ɤ-glutamyl hydroxymethyl rhodamine green fluorescence is a good postoperative prognostic indicator in patients with non-small cell lung cancer.