The invention discloses a method for applying multiple biological active agents to desulfurization of coking diesel oil. The method comprises steps as follows: (1), choline inositol composite powder or choline inositol composite pieces are added to the coking diesel oil, the mixture is heated and stirred by a magnetic stirring apparatus and react for 60-100 min, and coking diesel oil is taken out and cooled to the room temperature; (2), insulin is added to the coking diesel oil, and then the coking diesel oil is placed in a constant-temperature water bath environment and is heated and stirred at constant temperature; (3), a cholic acid active agent is added to the coking diesel oil, and the coking diesel oil is placed in the constant-temperature water bath environment and is heated and stirred at the constant temperature for 1-24 h; (4), a catalyst and an oxidizing agent are added to the coking diesel oil successively, and the coking diesel oil, the catalyst and the oxidizing agent have a catalytic oxidation reaction for 50-100 min; (5), an extraction agent is added in a volume ratio of the extraction agent to the coking diesel oil being 1:1, the mixture is stirred by the magnetic stirring apparatus for 5-10 min, poured into a separating funnel and left to stand for 20 min, and a diesel oil layer is collected. According to desulfurization refining with the method, the reaction conditions are mild, environmental protection is realized, the desulfurization rate is high, and the coking diesel oil has remarkable yield and is light in color and smell.