To study the effect of actual operational condition on the fatigue damage of lateral connection structure for fabricated concrete girder bridges, a fatigue damage assessment framework for the steel plate connection structure of this bridge type was presented based on linear Miner cumulative damage criterion. The influence rules of dynamic impact effects from stochastic traffic, traffic condition and progressive deterioration of bridge deck roughness on the fatigue damage of lateral connections were studied through an actual bridge example. The results show that the fatigue life of the transversal beam is obviously underestimated when considering the vehicular impact effect under Chinese specification compared with that of stochastic traffic with 95% confidence level. Traffic condition also has a significant influence on the fatigue damage, and the structural fatigue cumulative damage under intensive traffic condition is smaller than that of general one. Progressive deterioration of bridge deck condition has an obvious effect on the fatigue damage. The fatigue life of lateral connection decreased by 60.01% and 34.88% under general and intensive traffic conditions considering bridge deck degradation. When designing a new bridge, the impact factors from specification and general traffic condition should be considered in the fatigue calculation for lateral connection structure. It is suggested that considering the influence of stochastic traffic dynamic impact，operation traffic status and progressive deterioration of bridge deck was necessary when evaluating lateral connection fatigue damage for the existing bridges.