The invention discloses a tissue stem cell extract, a preparation method and an application thereof. The method comprises following steps of (1) centrifuging a mice tissue homogenate containing tissue stem cells and collecting a supernatant; (2) conducting a ConA agarose gel column chromatography to the supernatant and collecting an eluent; (3) conducting a Hitrap Q anion exchange column chromatography to the eluent and collecting the eluent; (4) concentrating the eluent to obtain the tissue stem cell extract. After an organism is immunized by the extract of the invention for a plurality of weeks, a specific immune response can be initiated for the organism, and in vivo tumor stem cells and/or tumor resting cells and cancerous cells can be eliminated, thereby achieving the aims of preventing and curing cancers. The extract of the invention can serve as a tumor preventive vaccine for preventing and curing autologous or allogenic tumors and for reducing the incidence of cancers among healthy people and can also serve as a tumor therapeutic vaccine for treating postoperative patients to effectively prevent metastasis and recurrence or serve as an aid therapy to chemotherapy.