The exceptionally low geothermal gradient (~2 °C/100 m) in the Tarim Basin, notably in the Shuntuoguole (STGL) area with depths exceeding 7000 m, has catalyzed the discovery of ultra-deep liquid hydrocarbons. Yet, the genesis of these hydrocarbons remains elusive, presenting significant challenges to further exploration and development endeavors. To address this quandary, an extensive analysis involving biomarker assessments and compound-specific δ2H and δ13C isotopic evaluations of n-alkanes was conducted, unveiling the secondary alterations and origins of these crude oils. For the first time, an approximately horizontal distribution of compound-specific δ2H and δ13C in n-alkanes was observed. Through the integration of diagnostic biomarker compounds, it was elucidated that the STGL crude oils did not undergo significant biodegradation, thermal alteration, or thermal sulfate reduction. Biomarker- and compound-specific C/H isotopic correlations suggest that the STGL crude oil predominantly originates from the Lower Cambrian calcareous shale, positing it as a potential end-member oil for this lithofacial source rock. Furthermore, the STGL crude oil exhibits similar biomarker configurations (notably abundant C28 triaromatic sterones, C23 tricyclic terpenoids, and aryl isoprenoids, with minimal 4-methylstane and gammacerane presence) to those oils previously considered as mixed from the Tazhong and Tabei Uplifts within the Tarim Basin. This similarity suggests that these previously deemed mixed oils may also derive from the Lower Cambrian calcareous shale. Consequently, more focused investigations into the Lower Cambrian calcareous shale are imperative to bolster refined deep and ultra-deep petroleum exploration efforts within the Tarim Basin in forthcoming studies.