In order to determine the asphalt to aggregate ratio range for rubberized asphalt based on the anti-fatigue property, ATB-25, the optimal rubber powder content was determined through the conventional asphalt test and dynamic shear rheological test (DSR). The initial asphalt to aggregate ratio was then established with the optimized Course Aggregate Void Filling method for ATB-25 mixture design. ATB-25 prism specimens were prepared to carry out four-point bending fatigue tests. Through these experiments, fatigue life equations were established for five different asphalt to aggregate ratios. Combining the flexural-tensile stress experienced by the ATB-25 based layer on a certain expressway in Hunan, the relationship curve between the flexural fatigue life of ATB-25 and asphalt to aggregate ratio at that stress level was obtained. Taking ATB-25 flexural fatigue life and shear fatigue life as criteria, the asphalt to aggregate ratio that meets the anti-fatigue performance requirements was determined to be 4.0%~4.4%. Results indicates that the rubberized asphalt ATB-25 mixture designed according to optimization asphalt-aggregate ratio method meets the requirements of anti-fatigue performance, and exhibits excellent high temperature stability.