A breakthrough has been made in the recent exploration of the deep oil and gas bearing system in the eastern belt around the Penyijingxi sag of the Junggar Basin. These reservoirs are characterized by mixed sources and multi-stage accumulation. However, this process has not been thoroughly investigated, limiting our understanding of the fundamental rules of hydrocarbon migration and accumulation and making it difficult to determine exploration plans. This study mainly reconstructs this process using biomarkers, carbon isotopes, light hydrocarbons, and fluid inclusions. According to the biomarkers and carbon isotopes for oil-source correlation, Permian crude oil is a mixed-source oil from the Fengcheng Formation (P(1)f) and the Xiawuerhe Formation (P(2)w) source rocks, while Jurassic crude oil originates from the P(2)w source rock. The carbon isotope and light hydrocarbon data demonstrate that Jurassic natural gas has a mixed-gas characteristic with a preponderance of coal-type gas, in contrast to Permian natural gas, which is primarily oil-type gas. The hydrocarbon charging events in the study area were reconstructed based on a comprehensive investigation of the hydrocarbon generation history of source rocks, the homogenization temperature of fluid inclusion, and the burial history of the reservoir. According to the model, the P(1)f and P(2)w source rocks have made contributions to the current regional oil reservoirs, which provides targets for future exploration.