The invention relates to a compound inhibiting angiogenesis which is obtained by extraction and separation from a fermentation culture of symbiotic microbe of sea cucumber, and a preparation method thereof. The preparation method comprises the following steps: firstly, by utilizing a PDA (potato dextrose agar) culture medium, the sea cucumber is cultured to get Alternaria sp. HS-3,2, a colony is white initially and gradually changed to be dark green, single black conidiophores on a matrix are in different length and has no branches on the top, and the conidiophores have vertical and horizontal diagrams and are obclavate and ovate; and secondly, a seed culture medium is produced so as to get a seed culture solution; and thirdly, the fermentation production is performed on the culture medium according to the proportion as follows: 0.5-1.5 parts of glucose, 0.05-0.2 part of yeast extract, 0.1-0.4 part of peptone, 1-1.5 parts of agar, 3-6 parts of sodium chloride and 100 parts of water; bacterial strains after well slant seed culture are selected into the fermentation culture medium and are standed for 1-2 months at the room temperature of 25-30 DEG C; and thalli of fermentation brothis removed by being filtered with gauze centrifugally, and supernatant fluid is concentrated at about 50-60 DEG C till 5L, extracted with ethyl acetate for 3-5 times, is separated by utilizing columnchromatography, and eluted with a petroleum ether and an ethyl acetate or chloroform and methanol flow system.