To quantity the comprehensive influence of both random and epistemic uncertainties during the process of flight vehicle stage separation, combined with the characteristics of hybrid model based on random and interval theory, a new reliability assessment method of separation between flight vehicle stages based on the hybrid model of random and interval theory was proposed. According to the requirements of the hypersonic vehicle separation mission, the separation kinetic simulation model was constructed. According to the geometric characteristic of the separation structure between the stages, a new rapid collision detection method was proposed. And the hybrid reliability assessment model of flight vehicle separation mission was constructed. The hybrid reliability assessment model was converted to unconstrained optimization problem of random reliability assessment. Considering of the characteristic of highly nonlinearity of system performance function due to complex external force and moment during the process of flight vehicle stage separation, the unconstrained optimization problem was efficiently solved by efficient global optimization and active learning Kriging method. It is shown that the influence of mixed uncertainty factors on the flight vehicle separation process can be described exactly through this method and the interval of flight vehicle separation mission reliability can be given accurately, which can further support the detailed design of flight vehicle separation.