In the present paper, the vibration response difference of the upper stage nozzle with higher expansion ratio between ground and altitude simulation hot-firing test is analyzed. It indicates that the acceleration response of the nozzle under ground hot-firing test is much higher than that of the altitude condition. In order to find the essential reason, the experimental and numerical simulation studies of the flow separation are developed by using the test engine nozzle. The experimental data show that the nozzle internal flow occurred flow separation and the divergence cone internal wall pressure pulsation increased significantly downstream from the separation location. The numerical simulation and experimental results indicate that the increase of internal wall pressure and turbulence pulsating pressure are the substantial reason of vibration response increasing aggravatingly during the ground firing test.