With the development of advanced and minimally invasive surgical techniques, and in view of the functional and cosmetic aspects, the need for rapid and accurate diagnosis during surgery is increasing. This study was conducted to develop a tissue diagnosis method using confocal microscopy after simple tissue staining that does not require freezing and slicing. At present, fluorescence staining with confocal microscopy is not generalized for real-time diagnosis during surgery. In this paper, we propose a fluorescence staining method using Hoechst 33342 and Eosin that does not require tissue freezing and slicing. The proposed method can be used as part of a rapid tissue diagnosis method that is suitable for use in the operating room, although further research is required before it can be applied in clinical practice.