The properties of indocyanine green (ICG) enable the observation of fluorescence images with a photodynamic eye (PDE) system after ICG is injected into a subject locally or intravenously. Over the last decade, many clinical applications using an ICG approach have been introduced, e.g., the detection of sentinel nodes in breast cancer and melanoma and the use of ICG as the contrast medium in cerebral angiography. However, an insufficient amount of basic research on the fluorescence properties of ICG has been done. In the present study, we first sought to identify the optimal ICG concentration that provides the maximum brightness fluorescence, in an in vitro experiment. The ICG solution used was 2.5 mg/10 mL of distilled water (original ICG solution). For additional diluted ICG solutions, we used physiological saline (saline) and distilled water. The results did not reveal the optimal concentration ratio of ICG and diluted solutions for obtaining the maximum fluorescence and intensity in each respective solution. In the next experiment, we added bovine albumin (2 g/dL) to each diluted solution. We then evaluated the appropriate dilution ratio of bovine albumin solution and ICG solution, and the maximum brightness of the ICG fluorescence was observed using the dilution of ICG solution to approx. 90-fold from 100-fold. In another experiment, the maximum intensity of ICG fluorescence was present in approx. 90-fold-diluted ICG solution in plasma. We further assessed the characteristics of ICG fluorescence in various conditions of temperature, pH, and light/dark. We observed no remarkable changes of ICG fluorescence intensity from 10 to 50 °C. Additionally, the ICG fluorescence was affected by the acid or alkali status and was preserved under the cold/dark condition.