The internal quality of watermelon is closely related to its vibration characteristics. As a representative of the non-contact vibration method, laser Doppler vibrometry technology can accurately measure the real vibration of agricultural tissue, so as to obtain the information of internal quality of the agricultural products. In this paper, the influence of vibration parameters on the watermelon vibration frequency response characteristics was firstly studied based on single factor experiments with the amplitude of acceleration, the frequency sweep rate and testing point of watermelon. Then, a interaction of multi-factor orthogonal experiment was conducted, which was performed by the laser Doppler vibrometry system based on the above factors and repeated three times at each parameter combination. The results of the single factor experiment indicated that the amplitude of acceleration and frequency sweep rate had significant effects on frequency spectrums, but the effect of testing point was not significant. The results of interaction of multi-factor orthogonal experiment showed that the optimal combination of the amplitude of acceleration, the frequency sweep rate and testing point for watermelon vibration measurement was 2.5 g , 1 000 Hz/min and the sunny side of the equator. The results laid a foundation for accurate nondestructive detection of internal quality for watermelon.