The invention discloses a formaldehyde-free animal specimen fixed liquid and a preparation method thereof, the formaldehyde-free animal specimen fixed liquid includes the following components: a biocide mildewcide, a protein denaturant, a polyol moisturizing agent and balance of water, and if the biocide mildewcide is in solid state, the mass concentration of the biocide mildewcide is 5-20 g/L, if the biocide mildewcide is liquid, the volume concentration of the biocide mildewcide is 1-2%; the volume concentration of the protein denaturant is 5%-40%; and the volume concentration of the polyol moisturizing agent is 2% to 10%. The formaldehyde-free animal specimen fixed liquid does not contain formaldehyde, and is non-volatile, and harmless to the human body; a fresh animal organ is washed with normal saline, and stored in the fixed liquid, a year later, the organ is not corrupted, free of mildew spot, and free of obvious color and shape change, the fixed liquid color has no obvious change, and the storage effect is good.