The invention provides an earthworm cast bio-fertilizer capable of controlling clubroot of cruciferae crops remarkably. The bio-fertilizer is prepared from components in parts by weight as follows: 1,000-1,200 parts of an earthworm cast and crop straw fermentation product and 1-3 parts of an antibacterial agent, wherein the weight ratio of the earthworm cast to crop straw is (5-8):1; the antibacterial agent contains at least one of Flowncide and Segway; pH of the bio-fertilizer is 6.8-7.2. The invention further provides a preparation method and an application method of the bio-fertilizer. The earthworm cast and the crop straw as two kinds of agricultural and breeding waste are taken as raw materials, and the cost is low; the formula and the preparation process of the bio-fertilizer are simple; the bio-fertilizer has a good control effect on clubroot of the cruciferae crops, and the control efficiency can reach 92%-93%; besides, the required application amount of the bio-fertilizer is small.