When the transmitted control signals in the forward channel are maliciously tampered by false data injection attacks (FDIAs), their authenticity is destroyed, leading to networked control systems (NCSs) performance losses or even instability. This paper proposes a Stackelberg game-based real-time defense strategy against FDIAs. First, if the transmitted control signals are tampered and the cost function of the attacker is not leaked, the defender makes the best response only based on its own cost function during each control cycle. Second, considering these sequential asymmetric information interactions between the attacker and defender, Stackelberg game is employed to establish the optimization model including cost functions and constraints. Third, Stackelberg equilibrium of the established optimization model is solved by extremum-based optimal strategy algorithm to obtain real-time defense strategy, while asymptotic stability of the system is proved. Finally, the feasibility and effectiveness of the proposed defense strategy are confirmed.