Tracing secondary oil migration pathways is critical for understanding petroleum system evolution histories. Traditional tools (e.g., molecular indicators and numerical modeling) utilized for evaluating oil migration processes either lead to ambiguous interpretations or only provide qualitative estimates. We quantitatively constrain secondary oil migration processes under an absolute time frame by integrating oil-inclusion fluorescence and in situ calcite U-Pb dating on calcite veins and cements hosting primary oil inclusions. Fluorescence spectra of oil inclusions and U-Pb ages were obtained on samples from ultra-deep Ordovician reservoirs along two major faults in the Halahatang oilfield, Tarim Basin (northwestern China). Absolute U-Pb ages suggest two main oil charge events during 475–433 Ma and 294–262 Ma, respectively, and revealed a northward-decreasing trend for oil maturity during single charge events. Vertical migration of oil from in situ source rock through active (or reactivated) faults is believed to be the key process inducing the spatial maturity variation in charged oils and considered as the main mechanism of secondary migration, with brecciated fault zones and dilatant fractures along faults acting as major vertical oil-migration pathways. The successful application of this approach has wider implications for elucidating petroleum migration processes in tectonic complex basins worldwide.