Abstract: The effect of long-term mixed application of organic manure and chemical fertilizers on farmland weed soil seedbank was evaluated in this study. The weed soil seed bank’s structure and biodiversity characteristics were studied by using the community ecology method under seven different fertilization treatments at soybean cultivating season of the wheat-soybean cropping system. The results show that all the weed seeds affiliated with sixteen species and ten families are detected, and the weed soil seed density is from 15995 to 106300 grain/m2. In the treatments with mixed organic manure and chemical fertilizers, the composition of weed species is relatively simple, and the number of the dominated species is from 1 to 2. In the treatment with mixed wheat straw and chemical fertilizers, the dominated species is Mollugo pentaphylla, and in the treatment with mixed manure and chemical fertilizers, the dominated species is Ammannia baccifera. The density of weed soil seedbank is reduced in the range from 15995 to 41900 grain/m2. The species richness is reduced in the range from 7.67 to 9.33, and the Shannon-Wiener index and Pielou index are in the range of those of the CK2 and NPK treatments (from 1.5 to 2.0 and from 0.6 to 0.8). Types and application amounts of applying organic fertilizers affect the weed species structure features significantly, and the influences of organic fertilizer types are greater than those of application amounts of wheat straw. The results of this paper show that the mixed application of organic manure and chemical fertilizers not only has beneficial to get high yield, but also regulates weed soil seedbank by adjusting the types and application amount of organic fertilizer to achieve economic and ecological benefits.