Abstract: The southern Ordos Basin has excellent petroleum exploration prospects. However, the tectono-thermal history and the controls on petroleum generation, accumulation and preservation conditions in southern basin are unclear. In this study, we analyzed the present geothermal field, paleo-geothermal gradient, maturity of the hydrocarbon source rocks, uplift and cooling process and tectono-thermal evolution history. In the study area, for the Ordovician, Permain and the Triassic strata, the present temperature is low (30−70 °C) in the southeastern area but high (80−140 °C) in the northwestern area. The paleo-geothermal gradient varied from 24 °C/km to 30 °C/km with a heat flow of 58–69 mW/m2 (i.e., a medium-temperature basin). The paleo-temperatures are higher than the present temperatures and the maximum paleo-temperatures controlled the thermal maturity of the source rocks. The vitrinite reflectance (Ro) values of the Triassic strata are >0.7% and the thermal maturity reached the middle-mature oil generation stage. The Ro values of the Permian-Ordovician strata are >1.8% and the thermal maturity reached the over-mature gas generation stage. The southern Ordos Basin has experienced the multiple tectonic events at the Late Ordovician Caledonian (452 Ma), Late Triassic (215 Ma), Late Jurassic (165−160 Ma), End-Early Cretaceous (110−100 Ma) and Cenozoic (since 40 Ma). A large-scale tectono-thermal event occurred at the End-Early Cretaceous (110−100 Ma), which was controlled by lithospheric extension, destruction and thinning. This event led to the highest paleo-temperatures and thermal maturities and coeval with the peak period of petroleum generation and accumulation. The southern Ordos Basin has undergone rapid and large-scale uplift since the Late Cretaceous due to expansion of the northeastern margin of the Tibetan Plateau, uplift of the Qinling orogenic belt and thrust faulting in the Liupanshan tectonic belt. The southern Ordos Basin experienced tectonic overprinting that was strong in the south and weak in the north. The strongest overprinting occurred in the southwestern part of the basin. The large-scale uplift, denudation and faulting led to oil and gas loss from reservoirs. The petroleum generation, accumulation and preservation in the southern Ordos Basin were affected by deep lithospheric structures and the tectono-thermal evolution. This work provides a novel tectono-thermal perspective on the petroleum generation, accumulation and preservation condition of the southern Ordos Basin.