Abstract: Fluorescence imaging now becomes an intraoperative navigation technique that gaining popularity in surgery and clinical research. However, at present, there is no mature and reliable method or other related guidance documents for the detection of fluorescence imaging performance. The performance analysis and quality supervision of products on the market could not be performed, which affects their clinical use and image quality. In this paper, a standard method of fluorescence imaging performance testing for fluorescence imaging system is proposed. Several kinds of fluorescence imaging performance parameters affecting fluorescence images are defined strictly. We also recommend scientific and feasible methods for their detections and analyses, which are verified by practical examples. This paper aims to provide a feasible reference standard for fluorescence performance evaluation.