Fluorescein sodium (FL) has been widely used in disease diagnosis and surgery. It is a yellow-dye that fluoresces when the light at wavelengths from 400 nm to 490 nm is irradiated, and its fluorescence can be observed with a surgical fluorescence microscope and a long-pass filter. However, even if an advanced microscope is used, it fails to produce a clear fluorescence image. As a result, it is possible that surgeons are experiencing difficulty in distinguishing tumors from blood vessels. Thus, this paper investigated the effects of a long-pass filter on the fluorescence image and discovered characteristics that affect the quality of the image. In this study, 0.0102mM of FL and a near-infrared camera were prepared, and the fluorescence was observed both in the presence and absence of a long-pass filter. By comparing two different types of the image, it was revealed that a filter is a necessary component in fluorescence microscopy as it blocks undesired light. Furthermore, the intensity of fluorescence after passing through a fabricated filter and a conventional filter was measured. It was discovered that a filter that transmitted the more light has higher transmittance (T) and lower power loss (PL) and optical density (OD). Given that an image becomes clearer if a filter transmits more light, it was concluded that a filter with higher T and lower PL and OD would produce a clearer fluorescence image. If this type of a filter is used in surgery, surgeons will be able to more effectively carry out tumor resection.