The invention discloses a pathological examination antiseptic hand cleaner and a preparation method thereof and belongs to the field of traditional Chinese medicine. The pathological examination antiseptic hand cleaner comprises the effective components including, Chinese starjasmine stem, clerodendrum bungei, sargent gloryvine, semen raphanin, sculellaria barbata, amaranth, pipewort, Chinese holly, herba epimedii, chinaroot greenbrier, wild chrysanthemum, acanthopanax root, honeysuckle, liquorice, radix asparagi, strobilus pini pine core, radix celastri orbiculati, ligusticum wallichii, cortex phellodendri, hibiscus syriacus, oldenlandia diffusa, sanguis draconis, aspongopus and borneol. The pathological examination antiseptic hand cleaner is made from pure Chinese herbs and achieves theeffects of clearing heat, removing toxicity, invigorating blood circulation, dredging collaterals, killing parasites, relieving itching and moisturizing skin, can effectives kill staphylococcus aureus, Escherichia coli, typhoid bacillus, pneumococcus, streptococcus mutans, pseudomonas aeruginosa, shigella dysenteriae and the like, and meanwhile, is high in bactericidal ability, free from skin irritation, safe, free from toxic and side effects as well as environmental pollution and accordingly applicable to generalized application.