The invention relates to cefathiamidine monocrystal, a preparation method, use and a composition thereof. The cefathiamidine monocrystal disclosed by the invention is prepared by comprising the following steps of: adding three solvents, namely ethanol, water and acetone, into a clean conical flask in any sequence according to any proportion, wherein the volume ratio of ethanol, water to acetone is (4-10):1:(0.05-3.67), during, before or after adding various solvents, adding cefathiamidine, dissolving at room temperature, and preparing cefathiamidine solution with the concentration of 0.05-0.5 g / ml; sealing by using a plastic film, and pricking a plurality of small holes on the plastic film; storing at 0-15 DEG C; and beginning to generate tiny crystals after about 48 h, and then obtaining cefathiamidine monocrystal after above 96 h. The preparation method disclosed by the invention is simple and convenient for operation and simple in equipment; the prepared monocrystal is good in stability; the cefathiamidine monocrystal composition disclosed by the invention is a composition composed of monocrystal and pharmaceutically acceptable carriers, preferably injection; the cefathiamidine monocrystal disclosed by the invention can be used for preparing medicines for treating respiratory tract infection, biliary tract infection, urinary tract infection, wound and surgical infection, skin and soft tissue infection, gynaecological infection, ear-nose-throat infection, endocarditis, blood poisoning, pneumonia and meningitis.