The fatigue performance of copper conductor is a key factor affecting the service life and reliability of flexible electronic equipment. Therefore, a quantitative test method for the fatigue performance of membrane  base structures is proposed. Firstly, the variation rule of viscoelastic parameters of copper wires with deposition thickness of 25 nm to 400 nm on flexible substrate was analyzed by using nanometer dynamic mechanical analysis technology, and the fatigue life of membrane  base structure was determined. Secondly, the microstructure of copper wires was observed and analyzed by scanning electron microscope (SEM) to explore the mechanism of wire thickness affecting the fatigue performance of membrane  base structure. Finally, the test results of copper wires with different thickness are analyzed and summarized to optimize the length and scale process design parameters of flexible electronic devices. The results show that the fatigue life of copper wires with the thickness of more than 100 nm is millions of times, the grain distribution is uniform, the average grain size is 50 nm, the stable distribution of the grain inhibits dislocation movement, and the fatigue performance is reliable. Therefore, copper wires with a thickness of 100 nm deposited on the flexible substrate have great advantages in improving the service life of flexible electronic devices.