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Biomarkers in source rocks are crucial in uncovering biological source and depositional environment. However, the detection of critical biomarkers such as steranes and terpanes in high-maturity source rocks is often challenging. On one hand, biomarkers in high-maturity source rocks are inherently present at low concentrations; on the other hand, the detection of these low-concentration biomarkers can be hampered by the high concentrations of interfering compounds, such as n-alkanes and other branched/cyclic alkanes. In this study, we developed a sample pretreatment protocol of stepwise extraction, using the combination of solvent in varying polarities (i.e., n-hexane and dichloromethane) and samples of different sizes (i.e., blocks >3 cm, 2.5–5 mesh, 10–20 mesh). The specific procedures are as follows: (1) removing the high amounts of interfering compounds during the first-step extraction to increase the relative proportion of biomarkers in saturated fraction by extracting coarse samples with relatively weak-polar solvents; (2) analyzing the soluble organic matter extracted in the second step by Gas chromatography-mass spectrometry (GC-MS) for biomarker (i.e., steranes and terpanes) detection by extracting samples of 100–200 mesh with solvents of stronger polarity. This method was demonstrated using organic-rich shales with high maturity (Ro = 1.42%) from the Qingshankou Formation (Fm.) in the Gulong Sag, and it was validated using the medium maturity (Ro = 1.0%) shales from the Qingshankou Fm., Sanzhao Sag, Songliao Basin. The results show that under routine Soxhlet extraction combining GC-MS analysis, sterane and terpane biomarkers in the Qingshankou shales are unidentifiable, regardless of whether urea adduction is applied or not. In contrast, these biomarkers can be successfully identified utilizing stepwise extraction coupled with GC-MS analysis. The effectiveness of biomarker identification is influenced by the interplay of both the shale particle size and organic solvent polarity. The integration of biomarker indexes was then employed to interpret the biological source and the depositional environment of the organic matter, providing detailed insights of the hydrocarbon generation potential of shales. This information can further provide additional guidance for selecting favorable resource zones for shale oil exploration in the Gulong Sag, Songliao Basin.