The aileron cabin is one of the important parts of civil aircraft. Its main function is to connect the aileron and provide support for the installation of the aileron, and transfer the aerodynamic load of the aileron to the main wing box. When subjected to aerodynamic forces and other excitations, the aileron cabin structure produces structural vibration response. The vibration fatigue life of the aileron cabin structure must meet the requirements of civil aircraft airworthiness clauses. In order to show compliance with the airworthiness clauses, it is of great significance to predict the vibration fatigue life of the aileron cabin structure under the excitation of random dynamic loads. Taking the aileron cabin structure of civil aircraft as the research object, an engineering treatment method suitable for vibration fatigue life prediction was proposed based on the measured strain data of flight test, the random vibration S-N curve of metal materials and the improved acoustic fatigue method. Through the combination of flight test, finite element simulation, data analysis, etc., the vibration fatigue life before and after optimization of the aileron cabin structure was predicted. Before the optimization of the aileron cabin structure, the predicted life of the damage location was less than 59 flight hours, which was close to the actual life. After the optimization of the aileron cabin structure, the stress level is significantly reduced, and the service life meets the requirements. The results show that the method for predicting the vibration fatigue life of the aileron cabin structure based on the measured data is effective and can be used as an engineering treatment method for the prediction of the vibration fatigue life.