Exploration potential is huge and the oil resources are rich in the Ordovician reservoirs of the Tarim Basin. However, the mechanism of hydrocarbon accumulation is complex and not yet fully understood. In the Tuoputai area, the hydrocarbon migration pathways and characteristics of deep hydrocarbon accumulation are revealed through analyses of the physical data of rich oil and gas, the geochemical parameters of oil, and fluid inclusions. The results show that the Ordovician oils in the Tuoputai area have the same geochemical characteristics as the mixed oil from the Lower Cambrian source rock and the Middle–Upper Ordovician source rock. The Ordovician reservoirs have been charged three times: in the late Caledonian, late Hercynian, and Himalayan stages. Oil charging occurred in the Hercynian stage, in particular, as it is the main filling period of hydrocarbon. The north-northeast (NNE)-trending TP12CX major fault, active in in these times and is dominant migration channel of hydrocarbon, but there is segmentation affected by the difference of activities. Oil maturity is higher in the south than in the north and is abnormally high near the major fault. Parameters related to migration indicate that oil migrated northeastward along the NNE-trending TP12CX major fault and adjusted laterally along the secondary faults and weathering crust, forming the present characteristics of oil and gas distribution.