Abstract: Molecular biomarker and whole oil carbon isotope (δ13Coil) analyses were conducted on eleven typical crude oils from the Qiongdongnan Basin to investigate their biological sources and depositional environments. Saturated hydrocarbon biomarkers in most samples are characterized by angiosperm-derived compounds, with aromatic compounds dominated by the naphthalene, phenanthrene, biphenyl, and fluorene series. The related source rocks of these oils were mainly deposited under oxic condition, but a subanoxic–suboxic and enclosed water column condition in the Central Depression during Oligocene. The identification of simonellite and related compounds in the aromatic fractions provides reliable evidence for the input of coniferous gymnosperms. Cadalene may also have a potential association with gymnosperms since it shows a strong positive correlation with simonellite. Evidence from density, n-alkanes, short-chain alkylbenzenes and secondary brine inclusions indicates that the unique crude oil B13-1 may have suffered from thermal alteration. These crude oils (excluding B13-1) can be classified into four types based on the δ13Coil values and molecular biomarkers. Type A oil (solely S34-3) is characterized by non-angiosperm plants, with minor dinoflagellates and increasing contribution from conifer gymnosperms than others. Type B oils (L17-2, L18-1, L25-1, and L25-1W) show heavy δ13Coil (−24 ‰ to −25 ‰) and mixed contributions from both angiosperms and marine algae, with the marine algae contribution increasing. Type C oils (L13-2 and B21-1) share similar biological sources with Type B, but the moderately δ13Coil (−25‰ to −26‰) and high level of terrestrial biomarkers suggesting a predominant contribution of angiosperms. Type D oils (sample Y13-1a, Y13-1b, and Y13-4) possess the lightest isotopes δ13Coil (mainly below −26‰) and are primarily derived from angiosperms, with mangrove vegetation playing an important role. Spearman correlation analysis among 14 source biomarker parameters with δ13Coil and geological setting of related source rocks implied that the marine algae should be responsible for the heavy δ13Coil in the Type B. The contribution of marine algae in the Central Depression may have been neglected in the past, as it is usually covered by remarkable angiosperm biomarkers.