The dynamic process of generation, expulsion, retention, and accumulation of shale oil within laminated shale has attracted considerable attention because it provides important geological insights into thermal evolution. Organic-rich tuffaceous laminated shale from the Chang 73 sub-member of the Ordos Basin provides an ideal case for investigating the micro-migration and accumulation of shale oil, which was investigated using high-temperature and high-pressure thermal simulation experiments and a series of organic geochemistry methods. The dynamic measurement results indicate that the hydrocarbon generation and expulsion processes of shale oil can be categorized into four stages: slow hydrocarbon generation stage (Stage A), slow hydrocarbon generation-expulsion stage (Stage B), rapid increase stage of massive hydrocarbon generation-expulsion (Stage C), and rapid decrease stage of massive hydrocarbon generation-expulsion (Stage D). A pronounced disparity between hydrocarbon generation and retained hydrocarbons is evident within the same laminaset during thermal stages, accompanied by dynamic functional transformations between the hydrocarbon-generating laminaset and hydrocarbon reservoir laminaset. This indicates that Rock-Eval pyrolysis parameters can effectively identify the micro-migration direction and quantity of shale oil based on the carbon balance principle. In early stages, hydrocarbons predominantly micro-migrate through connected pore throats within intra- or inter-laminasets, whereas in middle–late stages, fractures emerge as the primary micro-migration pathways. Light shale oil preferentially migrates and adsorbs onto mineral surfaces, whereas heavier components primarily migrate and accumulate along fractures. Free hydrocarbons are mainly hosted within intergranular and intragranular dissolution pores of tuffaceous laminaset, as well as in microfractures. The micro-migration and accumulation processes of shale oil in organic-rich tuffaceous shale are the result of the synergistic and dynamic evolution of the interactions between organic matter and inorganic minerals. These results provide enhanced insights into the differential micro-migration and accumulation mechanisms of shale oil within various lithological shales.