The invention discloses a preparation method of 5'-adenylic acid. The preparation method of 5'-adenylic acid comprises the following steps: performing synthesis reaction on powdered adenosine and polyphosphoric acid which serve as raw materials under the condition of no solvent, adding pure water after the synthesis reaction to perform hydrolysis, and refining the crude product obtained by the hydrolysis, wherein the mass ratio of the powdered adenosine to the polyphosphoric acid to the pure water is 1:(2-4):(2-4). The preparation method of 5'-adenylic acid has the following advantages: the solvent-free synthesis method is adopted, the reaction impurities are few, the condition is mild and pollution is avoided; the product yield is 90 percent or above, the product content is more than 99 percent and the conversion rate of the raw materials is high; and the operation steps are simple, the raw materials are wide in source and the price is moderate, so the preparation method of 5'-adenylic acid can be popularized and implemented on a large scale.