A Discrete Event System (DES) simulation and optimization model is proposed for the ambulance allocation problem in an Emergency Medical Service (EMS) system. The accuracy of the proposed model was improved by estimation of input parameters using the Poisson mixture model and the Expectation Maximization Algorithm. The results were proven by Mann–Whitney and Kolmogorov–Smirnov tests. The DES model was run by the OptQuest program embedded in Arena software. The response times of the cases within the urban and rural regions were improved by 40% and 45%, respectively, and the station success rates in urban and rural areas were raised to 95% and 96%, respectively. Our results indicated that the number of ambulances must be revised, and their allocations to stations in the real system need to be reorganized to achieve the required EMS system performance standards.