The invention discloses an anti-fatigue traditional Chinese medicine composition as well as a preparation method and application thereof. The composition is prepared from the following components in parts by weight: 13-62 parts of astragalus membranaceus, 48-102 parts of dried cuttlefish, 7-32 parts of radix paeoniae alba, 7-32 parts of fructus schisandrae, 3-17 parts of rhizoma cyperi, 7-32 parts of Chinese yam, 7-32 parts of epimedium, 10-52 parts of coix seeds and 1-17 parts of honey-fried licorice root. The traditional Chinese medicine composition has the effects of strengthening spleen, tonifying kidneys, regulating yin and yang of qi-blood along with soothing liver, so that five organs, yin-yang and qi-blood can be regulated. Clinical practice proves that the traditional Chinese medicine composition has an excellent anti-fatigue effect.