Combined microstructural, isotopic and chemical analysis of pyrite reveals the complexity of its genesis from the Jurassic Zhiluo Formation in the Diantou-Shuanglong sandstone-hosted uranium deposit, southern Ordos Basin. Framboidal, euhedral-subhedral and cement pyrite are identified by optical microscope and scanning electron microscope (SEM). Among them, euhedral-subhedral pyrite occurs in two forms, either as an independent mineral or wrapped by another phase of pyrite, where the wrapped euhedral core is designated as Py1, and another phase of pyrite named Py2 rims Py1. Sulfur isotope of pyrite by secondary ion mass spectrometry (SIMS) shows that each pyrite has a distinct composition (-30.64 %o to -17.94 %o for independent euhedral pyrite, -25.19 %o to +19.55 %o for cement pyrite, -4.79 to +3.73 %o for Py1, -24.27 to -10.15 %o for Py2). Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to reveal a considerable range of trace element compositions for different types of pyrite. The concentrated distribution of sulfur isotope values and the enrichment of trace element (Co, Tl, Sb, etc.) in Py1 indicate that it was formed by thermochemical sulfate reduction (TSR). Combined with relatively stable structures and absence of magmatic hydrothermal activity in the Ordos Basin, we propose that the rapid subsidence of the southern Ordos Basin in the Early Cretaceous led to increase of the basinal fluids temperature which reached the threshold temperature of TSR and formation of abiogenic pyrite. The negative sulfur isotope values and trace element depletion of other types of pyrite indicate that they were formed by bacterial sulfate reduction (BSR). Some cement pyrites show positive delta 34S due to Rayleigh isotope fractionation. Although the negative sulfur isotope values of Py2 indicates biogenesis, it contains relatively high trace element compared to other biogenic pyrite. At the end of the subsequent Early Cretaceous, uplift and denudation of the basin led to a decrease in geothermal gradient. Simultaneously, the Zhiluo Formation was exposed to the surface and received uranium-bearing oxidized fluids, indicating the beginning of uranium mineralization. Py1 of pre-ore pyrite was dissolved to form growth textures and release trace element during the infiltration of uranium-bearing oxidized fluids, and Py2 formed by the BSR has precipitated from trace element enriched fluids, resulting in moderately high trace element content and lighter sulfur isotope values. Therefore, uranium minerals precipitate around biogenic pyrite or between Py1 and Py2. We suggest that abiogenic redox processes and biogenic activities are involved in pyrite mineralization, which together promote uranium precipitation. This proposition is helpful for further understanding the mechanism of pyritization and uranium mineralization, and provides valuable guidance for thermogenic interpretation of other sedimentary basins with similar stable structures.