The invention discloses a preparation method of 1,4,9(11)-triene-androst-3,17-dione, which uses a compound I 9alpha-OH-4AD as an initial raw material and comprises the following steps: A) elimination reaction: adding the compound I into an eliminating solvent to eliminate 9- alpha hydroxy group by using the eliminating reagent, and reacting to obtain a compound II; and B) fermentation conversion: preparing a fermentation culture medium by using the compound II as a raw material, inoculating a Nocard's bacillus strain into the fermentation culture medium, culturing for conversion, and extracting to obtain a compound III, namely 1,4,9(11)-triene-androst-3,17-dione. The new initial raw material adopted by the method is cheap and accessible; and the new elimination method avoids ultralow temperature reaction, is simple to operate, and has the advantages of low equipment investment, fewer side reactions and high yield.