Fatigue failure is a fatal damage for the reinforced concrete structure bearing fatigue load repeatedly. That how to estimate or describe the degree of fatigue damage is a challenge in areas of structure damage and lifespan estimation. Structure's stiffness degenerates irreversibly along with the damage progress. There is a certain inherent relevance between stiffness degeneration and fatigue damage. A series of fatigue tests were carried out to study stiffness degeneration regulation due to its simplicity and feasibility. According to the test results, reinforced concrete beam's stiffness degeneration presents a three-stage rules obviously, and the stiffness degeneration curves accord with "S" style. Based on the stiffness degeneration regulation, the reinforced concrete beams' stiffness degeneration calculation formula is obtained by fitting experimental data. The formula has a perfect goodness of contact area with 10 test beams' experimental results, and it can describes reinforced concrete beams' stiffness degeneration perfectly. The formula can be used to forecast the deformation developing. Meanwhile, the residual life of the structure can be used to decision structure's fatigue fracture and the degree of performance degradation.