Abstract: Objective To develop a near-infrared fluorescence imaging system based on the fluorescence properties of methylene blue. Methods According to the optical properties of methylene blue, we used a custom-made specific LED light source and an interference filter, a CCD camera and other relevant components to construct the near-infrared fluorescence imaging system. We tested the signal-to-background ratio (SBR) of this imaging system for detecting methylene blue under different experimental conditions and analyzed the SBR in urine samples collected from 15 Wistar rats with intravenous injection of methylene blue at the doses of 0, 1.4, 1.6, 1.8, or 2.0 0 mg/kg methylene blue. Results The SBR of this imaging system for detecting methylene blue was affected by the concentration of methylene blue and the distance from the sample (P< 0.05). In the urine samples from Wistar rats, the SBR varied with the the injection dose, and the rats injected with 1.6 mg/kg methylene blue showed the highest SBR (8.71±0.20) in the urine (P<0.05). Conclusion This near-infrared fluorescence imaging system is useful for fluorescence detection of methylene blue and can be used for real-time recognition of ureters during abdominal surgery.