The second member of the Funing Formation in Qitong Depression, Subei Basin is a favorable section for shale oil exploration and development, where natural fractures provide important reservoir spaces and seepage channels for shale oil and have an important impact on shale oil exploitation and production increase. Based on core observation, imaging logging, thin section observation and experimental analysis, combined with advanced environmental scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM), this paper investigates into the genetic type, development characteristics, formation mechanism and distribution and occurrence state of natural fractures and their influence on oil and gas occurrence in the study area. The natural fractures are mainly tectonic fractures-including cross-bedding shear fractures, in-bedding shear fractures and in-bedding tensile fractures-and sedimentary rock fractures-mainly bedding fractures with a few sutures. The characteristics of fracture development in the sublayers of the second member vary greatly in the vertical direction. From sublayers Ⅰ to Ⅴ the fracture development intensity decreases and fracture types reduce to a single type; the fracture density is gradually reduced; the fracture extension length is gradually increased; and the fracture filling is gradually weakened. A complex fracture network is formed under various influencing factors including sedimentary rocks, structural setting and abnormal high pressure. The fracture network communicates with the reservoir matrix pores to form a three-dimensional pore-fracture system, providing a good space for shale oil storage and greatly improves reservoir permeability. As SEM/LSCM images show shale oil in the study area occur mainly as free or adsorbed oils in the forms of liquid oil droplets in pores and microfractures or oil films wrapping around mineral particles. The crude oil in fractures is mainly composed of light components, and the pores contain both light and heavy components. The development of natural fractures obviously improves the physical properties of shale reservoir rocks and is conducive to the later fracture network reconstructure—an important guarantee of shale oil enrichment and high-production in the study area.