The invention discloses fresh Cordyceps extract with anti-melanoma activity, preparation method thereof and application thereof in the antitumor field. The fresh Cordyceps extract is prepared via the steps of shearing fresh Cordyceps, adding water, homogenizing, subjected homogenized fresh Cordyceps to low-temperature ultrasonic assisted extraction, centrifuging, collecting supernate, and lyophilizing to obtain the fresh Cordyceps extract. The fresh Cordyceps extract prepared herein has good inhibitory effect for melanoma cells, is high in safety and has good medicinal prospect. The preparation process of the extract herein is simple, and the extract is easy to produce on large scale and safe to use.