The flexural fatigue performance of manufactured sand concrete(MSC) and river sand concrete(RSC) under constant amplitude of flexural cyclic loading at 5different stress levels was investigated. The two parameter Weibull distribution and S N Pf flexural fatigue equation were established and analyzed. Furthermore, the Micro analysis on the interfacial transition zone(ITZ) of MSC and RSC was carried out. The results show that the flexural fatigue lives of MSC and RSC follow the two parameter Weibull distribution and the distributions of flexural fatigue lives for MSC exhibit lower variability as compared to that of RSC. At the same stress level, MSC exhibits better flexural fatigue performance than RSC and the flexural fatigue lives of MSC are less sensitive to the change of the stress level than that of RSC. The 2×106cycles endurance limit for MSC is calculated to be 55%, which is 2% higher than that of RSC. The ITZ of MSC has a denser structure and higher micro hardness than that of RSC.