The invention relates to a novel acetylene compound applied to an anti-cancer drug as well as a preparation method and application thereof. The invention provides an acetylene compound of which the structural formula is as shown in the specification, wherein R represents a functional group; R=R, CR1R2R3, (CR1R2)nX or NR1R2; R, R1, R2 and R3 are hydrogen, halogen, C1-C24 alkyl chains, an aromatic group and a heterocyclic group; n is 1-6; X is amino, an ether group, an amidogent, sulfydryl, phosphino, halogen or an ester group; the aromatic group is phenyl, naphthyl, pyrenyl, anthryl, alkyl or halogen; the heterocyclic group is pyrrole, thiophene, pyridine, piperazine, pyran, furfuran, imidazole, parazole or morpholine. The experiment proves that the acetylene can lead obvious chain interruption of DNA and apoptosis of tumor cells. The acetylene compound proves that the synthetic acetylene has high biological activity.