Understanding multiscale migration differentiation within shale oil systems is critical for identifying high-mobility migration sweet spots that facilitate the sustainable shale oil recovery. However, owing to geological complexity and technical uncertainty, the chemical differentiation and mobility effects of shale oil migration have not been clarified. This study is focused on elucidating the μm–m-scale migration differentiation and mobility effects in shale oil systems by combining multiple methods, including laser scanning confocal microscopy (LSCM), nuclear magnetic resonance (NMR), multi-temperature pyrolysis, and extract geochemical analysis. Core samples collected nearly equidistantly from the Triassic Chang 7 Member shale system in the Ordos Basin are employed for investigation. The LSCM results reveal that interlaminar shale oil migration on the μm–mm scale manifests in the form of light/heavy component differentiation. However, cm–m-scale migration across laminae assemblages (or lithofacies) corresponds to pyrolysis parameter anomalies, original–true hydrocarbon generation potential differences, chemical compositions and n-alkane differentiation. The migration hydrocarbon inputs can improve the in situ oil composition and movable oil content, thus affecting shale oil mobility. The mobility disparities between source–reservoir units in a shale oil system are primarily controlled by the degree of migration differentiation. Notably, the good source–good reservoir laminated units at the μm–mm scale and the thick source–thick reservoir configurations at the cm–m scale exhibit relatively strong migration differentiation and favorable mobility improvement. These mobility patterns associated with μm–m migration differentiation are critical for evaluating high-mobility sweet spots and developing cost-effective and sustainable shale oil systems.