The invention relates to a preparation method of a compound reacting with alkaline phosphatase to create chemiluminiscence. The preparation method comprises the following synthesizing steps in sequence: (1) synthesizing acridine-9-sulfuryl chloride; (2) synthesizing 9, 10-acridanyl-9-sulfuryl chloride; (3) synthesizing 10-methyl-9, 10-acridanyl-9-sulfuryl chloride; (4) synthesizing 10-methyl-9, 10-acridanyl-9-thiocarboxylic acid-3'-ethylpatate; (5) synthesizing 9-(3'-phenetyl sulfurized phosphoricoxymethylene)-10-methyl-9, 10-acridanyl bi(cyanoethyl) ester. The preparation method is simple in technology, high in stability, and low in price.