The invention discloses an anti-attenuation fungal fluorescent dyeing liquid and a preparation method thereof. Wherein the components of the anti-attenuation fungal fluorescent dyeing liquid include:a fluorescent agent, a solubilizing agent, a penetrating agent, a background dye, an anti-fluorescent attenuation agent, a stabilizer, and deionized water. According to the anti-attenuation fungal fluorescent dyeing liquid and the preparation method thereof, each component is stabilized in a solution system, and filtered by a cellulose membrane, all kinds of minute impurities are effectively removed, thereby having an effect of long-term storage without crystallization and having the advantages that the fungal fluorescence mark and the background dyeing are synchronously carried out in a few seconds; the anti-attenuation fungal fluorescent dyeing liquid does not need to be operated in a dark room, the performance is relatively stable, and has the characteristics of convenient operation, short dyeing time, long attenuation time, high efficiency, comprehensive use range, high observation clarity, good specificity and accurate detection result, and has a good clinical application prospect.