Chitooligosaccharides (COS) significantly attenuates liver dysfunction. However, the conundrum of the oral bioavailability of COS limits their pharmacological effects. Therefore, a strategy of nanoencapsulation was employed to enhance oral bioavailability and tissue-targeted distribution of COS. In this study, nanospheres loaded with COS (CANs) were prepared, their bioavailability, biodistribution, transport mechanism and anti-liver fibrosis effects were explored. Nanoencapsulation improved the oral bioavailability of various COS monomers through microfold cell-mediated absorption route in an indiscriminate manner. CANs were more favorably enriched and protractedly accumulated in the liver. In a liver fibrosis model, CANs ameliorated the pathological state and extracellular matrix deposition. The alleviation of liver fibrosis for COS could be attributed to the inhibition of liver cell apoptosis and liver sinusoidal endothelial cell (LSEC) capillarization. Consequently, this study highlights the improved oral bioavailability of COS and proposes a novel mechanism of COS, for better understanding its hepatoprotective effect.