The invention belongs to the field of pharmaceutical chemistry and discloses Jaspine B and a 3-epi-Jaspine B azo analogue with anti-tumor activity, a preparation method and an application thereof. A target product is simply and quickly obtained through a two-step reaction. The target product has the following structural formula as shown in the description, wherein n is 6, 7, 8, 9, 10, 11 or 13. An in-vitro antitumor activity experiment verifies that the compounds have obvious inhibiting and killing effects on various tumor cells and provide screening drugs for development of a novel anti-tumor drug. An initial mechanism research verifies that the compounds can accelerate increase of expression quantity of autophagy marker proteins and reduce the potentials of mitochondrial membranes, explaining that the mechanism of action of the compounds is that autophagy is induced by the tumor cells.