The invention particularly relates to a bio-organic fertilizer containing bacillus pumilus. The bio-organic fertilizer containing bacillus pumilus is prepared from the following components in percentage by weight: 1%-5% of bacillus pumilus, 35%-40% of fuming slag, 35%-45% of peat, 8%-10% of an oil cake and 7%-12% of flower slag, preferably 1% of bacillus pumilus, 40% of fuming slag, 40% of peat, 10% of an oil cake and 9% of flower slag. A preparation method of the bio-organic fertilizer containing bacillus pumilus comprises the following steps: uniformly mixing the raw materials, then, adding water which accounts for 10% of the total volume of the material mixture and piling up for fermentation; and turning the pile once every five days, and fermenting the pile for 40 days to prepare the bio-organic fertilizer. The bio-organic fertilizer is applied as a base fertilizer, and a bacillus pumilus fermented liquid is diluted by 5000 times at the same time; the bio-organic fertilizer is irrigated to the roots of cigarette plants which are transplanted or sprayed to the leaves of the cigarette plants, so that the cigarette root knot nematode disease can be effectively prevented and controlled, and the environmental pollution caused by use of chemical agents is reduced.