The invention discloses a method of single time measurement of atractylenolide and atractylon. The procedure includes preparing mobile phase resolution A and B made of chromatographic pure methanol, acetonitrile and redistilled water, preparing the mixed control solution made of atractylenolide I, atractylenolide III and atractylon, preparing sample solution, testing the three absorption peaks of control solution and samples at three different time respectively by chromatograph of liquid according to the setting gradient of mobile phase A and B and the time, and calculating the content of atractylenolide I, atractylenolide III and atractylon according to the formula X=(peak area of sample is multiplied by density of control then is divided by peak area of control and is multiplied by density of sample) is multiplied by 100 percent. The invention can determine the content of atractylenolide I, atractylenolide III and atractylon in atractylodes macrocephala by only one sample injection. Thus the operation is simplified, the error of multiple sample injection is reduced and analysis of the constituent ration of the three ingredients is convenient. The invention is of important academic value and meaningful in practical application.