Since the heating substation plays a key role in transferring the thermal energy from the primary network to the secondary network and controlling the heat output of district heating system to meet the thermal load, high operation performance of heating substation is essential for energy conservation, cost saving and emission reduction. The dynamic operation stability of heating substation is a very important dynamic characteristic of heating substation and largely affects the operation efficiency of district heating system. The operation instability of heating substation mainly manifest as flow rate and pressure oscillations, which will deteriorate the network hydraulic condition, break the network thermal balance, reduce the consumer comfort and increase the energy cost of the pumping system. Since heating substations will easily operate unstably under some conditions, this paper presents a theoretical method to analyze the stability and retune the feedback controller for operation stability of heating substation. Mathematical model of the plate heat exchanger was established and the feedback control theory was adopted to study the operation stability of heating substation. Based on the mathematical model and feedback control theory, a stability criterion was proposed for analyzing the operation stability of district heating substation effectively. The dynamic model of plate heat exchanger was validated with measured data. Simulation results show that controller tuned at certain operating condition can’t ensure operation stability of heating substation, when operating condition varies in large range. The stability analysis method proposed in this paper can be applied to analyzing the operation stability and tuning the controller of heating substation to enhance the operation stability.