To improve the simulation accuracy of the Hardware-in-loop Simulation (HILS) of a guidance and control system, an idea is proposed to well balance the relationship between calculation ability and simulation accuracy of the system by selecting reasonable sampling steps and optimizing real time ability of the system.A mode for the HILS of guidance and control system is established, and the restraining conditions for sampling steps of the HILS are deduced according to the sampling principle.Furthermore, the restraining conditions are optimized and the optimized sampling steps are determined to implement the restraining conditions of calculation accuracy and stability.Finally, a HILS is carried out for the guidance and control system.In the simulation, the aircraft six Degree-of-freedom ( 6DOF) differential equation is written by C MEX S-function, and the hardware consists of a three axis turntable , a real-time calculator and a flight-motion-simulator.It is concluded that the HILS can meet the requirement of guidance and control systems for the simulation accuracy and real time stability in an optimized sampling step of 1 ms and can improve the simulation accuracy without adding any software or hardware.