Application of indocyanine green (ICG) fluorescence has led to new developments in gastrointestinal surgery. However, little is known about the use of ICG for the diagnosis of postoperative gut leakage (GL). In addition, there is a lack of rapid and intuitive methods to definitively diagnose postoperative GL. To investigate the effect of ICG in the diagnosis of anastomotic leakage in a surgical rat GL model and evaluate its diagnostic value in colorectal surgery patients. Sixteen rats were divided into two groups: GL group (n = 8) and sham group (n = 8). Approximately 0.5 mL of ICG (2.5 mg/mL) was intravenously injected postoperatively. The peritoneal fluid was collected for the fluorescence test at 24 and 48 h. Six patients with rectal cancer who had undergone laparoscopic rectal cancer resection plus enterostomies were injected with 10 mL of ICG (2.5 mg/mL) on postoperative day 1. Their ostomy fluids were collected 24 h after ICG injection to identify the possibility of the ICG excreting from the peripheral veins to the enterostomy stoma. Participants who had undergone colectomy or rectal cancer resection were enrolled in the diagnostic test. The peritoneal fluids from drainage were collected 24 h after ICG injection. The ICG fluorescence test was conducted using OptoMedic endoscopy along with a near-infrared fluorescent imaging system. The peritoneal fluids from the GL group showed ICG-dependent green fluorescence in contrast to the sham group. Six samples of ostomy fluids showed green fluorescence, indicating the possibility of ICG excreting from the peripheral veins to the enterostomy stoma in patients. The peritoneal fluid ICG test exhibited a sensitivity of 100% and a specificity of 83.3% for the diagnosis of GL. The positive predictive value was 71.4%, while the negative predictive value was 100%. The likelihood ratios were 6.0 for a positive test result and 0 for a negative result. The postoperative ICG test in a drainage tube is a valuable and simple technique for the diagnosis of GL. Hence, it should be employed in clinical settings in patients with suspected GL.