The invention discloses a preparation method of an isosorbide mononitrate injection, belonging to the technical field of pharmaceutical preparations. According to the preparation method, isosorbide mononitrate, propylene glycol, sodium acetate and 1% glacial acetic acid are taken as raw and auxiliary materials. The preparation method comprises the following steps: (1) weighing isosorbide mononitrate, propylene glycol, sodium acetate, glacial acetic acid and medicinal carbon; (2) pre-dissolving sodium acetate, adding 60% of prescription amount of injection water, and adding glacial acetic acid and propylene glycol; (3) pre-dissolving isosorbide mononitrate, uniformly stirring, adding the solution obtained in the step (2); (4) adding medicinal carbon into the solution obtained in the step (3), supplementing injection water to a full amount, standing, maintaining the temperature, cooling, filtering for decarburization, and detecting an intermediate product; and (5) encapsulating the intermediate product, and carrying out sterilization at 121 DEG C for 10-15 minutes, so as to obtain the isosorbide mononitrate injection. The preparation method has the beneficial effects that the process is simple, the increase of nitrite content is inhibited during the sterilization, and the clinical safety is effectively improved.