A microorganism crude oil decalcification agent and a preparation method and decalcification method thereof relate to the microorganism crude oil decalcification agent and the preparation method and decalcification method thereof. The components comprise the component A of oilfield produced fluid, the component B of waste molasses, the adding quantity of which is 2 percent to 6 percent of the weight of the component A, the component C of beef extract, the adding quantity of which is that each gram of crude oil produced fluid contains the component A with the weight of 3000 to 10000 microgrammes, and the component D of inorganic salt type micronutrient required by microorganism for growing, the adding quantity of which is that each kilogram of the oilfield produced fluid contains 1 to 1.5g of ammonium salt, 1.5 to 4g of sodium salt, 1 to 2g of potassium salt and 0.2 to 0.5g of magnesium salt. The crude oil decalcification method provided by the present invention is applied to the crude oil with any calcium content. The method has simple operation, low cost, high decalcification rate and good stability of the decalcification agent and is especially applicable to heavy crude oil, the calcium content of which is higher than 200 microgrammes/g.