According to the characteristics of periodic variable load in highway bridges, the calculation method of fatigue stress amplitude of steel structure of highway bridge is given according to the current "General Code for Highway Bridge and Culvert Design" (JTG D62-2004). According to the difference in structural importance and replaceability, according to the whole life design idea, on the basis of long-term and extensive tests of the Research Institute of Science and Technology of the Ministry of Railways, a fatigue resistance equation with the guaranteed rate of allowable stress amplitude as a variable is proposed, so as to achieve the purpose of comprehensively considering the entire life of construction and bridge service during the design. Taking the fatigue calculation of a steel mixed stiffener beam of a highway suspension bridge as an example, the reliability of the proposed method is proved by comparison with other normative methods.