The invention discloses a rapid assembling method of multi-fragment DNA yeast. The rapid assembling method comprises the following steps: (1) performing co-transformation on a plurality of DNA molecules with homologous arms, and linearized yeast shuttle vectors, so as to obtain yeast; (2) eluting all the transformed yeast colonies on a whole screening culture plate, centrifuging, and discarding the supernate to obtain transformed yeast cells; (3) extracting plasmid DNA of the yeast cells obtained in step (2), transforming the competent cells of colon bacillus; and (4) screening colon bacillus, and cloning to obtain large-fragment DNA. The method for assembling large-fragment DNA molecules is fast in speed, simple, convenient and feasible, high in success rate, low in cost, high in efficiency, easy to operate, beneficial in enlarging the industrialization scale and wide in application, and a plurality of small-fragment DNA molecules can be assembled into one large-fragment DNA molecule.