Objective: We study the biomedical optical properties of the color light and near-infrared fluorescence separated-merged imager. Materials and Methods: The color light and near-infrared fluorescence separated-merged imager can illuminate the visible light and the near-infrared light of 760 ± 10 nm, receiving the reflected light and 835 ± 10 nm near-infrared fluorescence, and display the color, fluorescence and merge image. ICG solution of different concentration, including standing time, was allocated to study the best imaging condition in vitro, and the depth of fluorescence penetration was studied with 5% agarose gel; the imaging characteristics of the imager was studied using SD rat; and then the SLNs tracing in 4 cases of penile carcinoma was performed. Results: When the concentration of ICG is 13.11 μmol/L, the fluorescence intensity and the merge image are the best. The maximum depth of fluorescence imaging is 9mm in 5% agarose gel, while the bone has the greatest influence on it. The SLNs tracing shows that the imager can locate the SLNs in vitro, to achieve perioperative navigation during biopsy. Conclusion: There are many factors that affect the imaging effect, but the imaging effect of the imager meets the requirement of vision in a wide range, and can effectively trace the SLNs in perioperative period.