Sediments with organic matter content (total organic carbon) TOC ≤ 0.5% which can act as effective source rocks are critical and challenging in the field of petroleum geology. A new method is proposed through a case study to identify and evaluate the effective source rocks, which is applied to study the changing characteristic of hydrocarbon-generation potential index with depth. The burial depth corresponding to the beginning of hydrocarbon-generation potential index reduction represents the hydrocarbon expulsion threshold in source rocks. Then, new identification standards are established for discrimination of effective source rocks of Middle–Upper Ordovician in Tarim Basin. The critical value of TOC for effective source rocks change with their burial depth: the TOC > 0.5% with source rock depth > 4000 m, TOC > 0.4% with depth >4500 m, TOC > 0.3% with depth > 5000 m, TOC > 0.2% with depth >5500 m. Based on the new criteria, effective source rocks in the Middle–Upper Ordovician are identified and their total potential hydrocarbon resources is evaluated, reaches 0.68 × 109 t in the Tazhong area, which is 65.4% higher than that of previous studies and consistent with the exploration result. Thus, this new method is of significance to resource evaluation and can be applied in the carbonate source rocks and mudstone source rocks with high degree of exploration.