The invention discloses a preparation method of 2,3-pentanedione. The preparation method comprises the following steps: one or two of 3-hydroxy-2-pentanone and 2-hydroxy-3-pentanone is/are added to water and uniformly mixed with water, ozone is introduced at the temperature of 3-20 DEG C for a reaction, and 2,3-pentanedione is obtained. According to the synthesis process, ozone is adopted to oxidize the mixture containing 3-hydroxy-2-pentanone and 2-hydroxy-3-pentanone, acetic acid is adopted as a cocatalyst, reaction conditions are mild, and operation process is simple; product yield is high;cost is low; the method has the advantages of being safe and environmentally friendly, and no wastewater is produced.