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In order to study the pressure fluctuation characteristics in the mixedflow pump caused by the effect of impeller and guide vane’s unsteady periodical interference, the pressure fluctuation in key monitoring points was measured on the mixed-flow pump body. The time domain and frequency domain responses of the pressure fluctuation under different flow rate conditions on the impeller inlet, middle part of impeller, impeller outlet, guide vane inlet, guide vane outlet and device exit were analyzed. The results showed that the impeller rotational cycle greatly influenced the pressure fluctuation at the positions of the impeller inlet and the blade rim central point of the impeller. The four peaks and four valleys were appeared in the fluctuation curves which were consistent with the number of impeller blades. Meanwhile, the main pressure fluctuation frequency was consistent with the impeller blade passing frequency. The pressure fluctuations appeared 7~12 peaks and valleys respectively in impeller outlet monitoring points and guide vane inlet monitoring points under small flow rate condition, which was caused by the rotor-stator interaction between impeller and guide vane. Besides, due to the rotor-stator interaction, the pressure fluctuation spectrum range became larger and frequency component was increased. The main frequency was shifted to high frequency with the decrease of flow rate. The fluid vibration induced by dynamic and static interference and the noise of high frequency composition was increased gradually. The pressure fluctuation amplitude of the monitoring points in the center of the impeller was the largest and the most sensitive to the change of flow rate, the pressure fluctuation of the monitoring points away from the impeller area was less affected by the change of flow rate. The research results had significant reference value for revealing the unsteady flow characteristics of mixed-flow pump.