This paper aims to separate a water-in-oil emulsion using ultrasonic standing waves generated by a piezoelectric transducer inside a channel under the transient flow regimes. In the proposed method, the acoustophoretic force at pressure nodes accumulates the water droplets pulled down due to gravity as they agglomerate and get heavy enough. Furthermore, a simulation was conducted to clarify and explain the fundamentals of the procedure. This approach was used at various flow rates (18, 26, and 34 ml/s) and different percentages of water-in-oil (20%, 40%, and 60%). According to the design of experiments based on response surface methodology (RSM), these parameters and their interaction are significant. Results indicated that the water separation was achieved successfully in a noticeable number of the conducted experiments which could confirm the proficiency of the proposed technique. The maximum separation percentage of 80 was obtained when the flow rate and water content were 18 ml/s and 20%, respectively.