Summary: This paper studies the \(M/G/1\) queueing system with startup time, bi-level threshold \((m,N)\)-policy and single server vacation without interruption. In this system, when the server is transferred on vacation, the server starts the system immediately if the number of waiting customers is no less than a given positive integer threshold \(m (m\ge 1)\), and when the system startup is complete, the server begins service immediately if the number of waiting customers is no less than another given positive integer threshold \(N (N\geq m)\). Assume that the server’s vacation time and the startup time of the system follow general distributions, both the transient queue-length distribution and the steady-state queue-length distribution of the system are discussed by using the renewal process theory, the total probability decomposition technique and Laplace transform tool. The expressions of the Laplace transformation of the transient queue-length distribution with respect to time \(t\) are obtained. Furthermore, the recursive expressions of the steady-state queue-length distribution are derived by a direct calculation. Meanwhile, the stochastic decomposition structure of the steady-state queue-length and the explicit expression of the additional queue-length distribution are presented. Finally, the explicit expression of the long-run expected cost per unit time is derived under a given cost model. And the numerical example is given to determine the optimal control policy \((m^*,N^*)\) for minimizing the long-run expected cost per unit time.