Organic matter is the basis of shale gas generation, and the study of Quaternary Pleistocene climate change in the Qaidam Basin and its effect on organic matter enrichment is crucial for the exploration and development of biogenic gas in the Qaidam Basin. In this paper, the Quaternary shale in the Qaidam Basin is taken as the research object, and Quaternary Pleistocene climate change is clarified in terms of paleo-moisture and paleo-temperature through organic carbon analysis and main and trace element experiments. Then, the influence of climate change on organic matter enrichment is analyzed from two perspectives: biological productivity and organic matter preservation. Finally, the Quaternary Pleistocene organic matter depositional pattern of the Qaidam Basin is established. The results show that (1) in the early-middle Quaternary Pleistocene, the climate was cool and humid, the herbaceous plants were luxuriant, and rich in cellulose, hemicellulose, sugar, starch and pectin, which improved the biological productivity of the surface layer of the water column. The amount of precipitation was high, and the stratification of the water column was good. The strong stratification of the water column also enhanced the reduction level of the lower layer of the water column, which is favorable for the preservation of the organic matter deposited from the upper layer and thus favors the enrichment of sedimentary organic matter. Additionally, relatively low temperatures inhibit the activities of methanogenic bacteria, which is also conducive to the preservation of organic matter. (2) In the late Pleistocene, under the Neotectonic Movement, the Tibetan Plateau uplifted, the climate became arid, and the temperature increased, leading to an increase in the proportion of woody plants and a decrease in the amount of nutrients available to methanogenic bacteria, decreasing the biological productivity of the surface layer of the water column. On the other hand, the stratification of the water column was weakened. The mixing of oxygen-rich water in the upper layer and oxygen-poor water in the lower layer results in the level of reduction of the lower layer of the water column being significantly lowered. The sedimentary organic matter that settled from the upper layer was easily destroyed, which was unfavorable for the preservation of sedimentary organic matter. Additionally, when the temperature was relatively high, methanogenic bacteria consumed a large amount of organic matter, which was also unfavorable for the preservation of organic matter. The research results have important theoretical and practical significance for the exploration and development of biogenic gas in the study area.