A large number of faults are developed in the southwest of Ordos Basin. The determination of fault activity time is very important to clarify its relationship with oil and gas accumulation. However, there is no definite understanding of these fault activity periods. Based on field investigation and three-dimensional seismic data analysis, this paper analyzes the NEE-trending faults developed in the Jurassic-Cretaceous of the Mesozoic, and uses the laser in-situ U-Pb dating method of calcite veins in the fault zone to analyze the fault activity time. The results show that the two ages of the syntectonic calcite veins in the NEE-trending fault are (125.3±1.9)Ma and (79.04±0.68)Ma, respectively.Combined with seismic data interpretation, it is comprehensively limited that the fault is mainly developed in the late Early Cretaceous-Late Cretaceous. The age of the two-stage fault activity corresponds to the two overall uplift time (170-120Ma) and (80-60Ma) of the Ordos Basin since the Early Cretaceous, indicating that the multi-stage fault activity and the overall uplift since the Early Cretaceous have a corresponding relationship. The time of NEE-trending fault activity corresponds to the main accumulation period of early Cretaceous oil and gas in the study area and the adjustment period of late Cretaceous reservoir. The influence of NEE-trending fault activity on oil and gas accumulation is mainly reflected in the communication between source kitchen and reservoir and the transformation and adjustment of primary reservoir. According to the quantitative calculation results of formation pressure relief caused by the overall uplift of the study area, the development of these faults is also an important reason for the abnormal low pressure of the reservoir in the basin.