Unsteady separated flow is a common flow condition causing many detrimental effects in aerospace and other fields. Open-loop control is a potential means to eliminate these drawbacks. At present, the unsatisfactory performance of open-loop control mainly attributes to the high-dimensional parameter optimization procedure and the lack of efficient knowledge-guided control law design methods. This paper proposes a method managing to directly obtain the anti-resonant frequency from the input and output amplitude-frequency characteristic curves of the reduced-order model of the flow-control coupled system. Simultaneously, a negative gain criterion is proposed to judge whether the target control effect can be achieved under the current parameter combination. For the control of low Reynolds number flow around a cylinder, the simulation results show that the optimal open-loop control frequency is 1.268 times the natural frequency of the flow, which is consistent with the anti-resonant frequency, and 26.8% of the lift fluctuation is suppressed. This paper also studies the influence of parameters such as flow frequency change, control start time, jet mass flow rate, and so on. Furthermore, control position is the key parameter affecting the amplitude-frequency characteristics. The anti-resonance points on the curves corresponding to different control positions can also guide the design of the optimal control frequency, and the negative gain criterion is still applicable. This method greatly reduces the time consumption in parameter optimization and improves the engineering application prospect of the open-loop control in unsteady separated flow control.