To study the influence of prestressing on distributed feedback (DFB) fiber laser hydrophone in underwater acoustic detection, a theoretical model of DFB fiber laser hydrophone with two diaphragms, which presents the relation between pressure sensitivity and prestressing force, is established. The relation between dynamic performance of hydrophone and prestressing force is simulated and analyzed by finite element software ANSYS. Prototypes of the DFB fiber laser hydrophones under different prestressing forces are fabricated and tested in an anechoic tank. The experimental results show that the frequency response of hydrophone becomes flatter with the increasing of prestressing force. When the wavelength variation induced by prestressing force increases to 400 pm, the acoustic pressure sensitivity of hydrophone is 136.8 dB±0.3 dB in a frequency range from 2.5 kHz to 10 kHz. However, the frequency response curve changes small when prestressing force continues to increase, which agrees well with both the theoretical analysis and simulation results.