Because of stem cells are limited by the low efficiency of their cell homing and survival in vivo, cell delivery systems and scaffolds have attracted a great deal of attention for stem cells’ successful clinical practice. β-chitin nanofibers (β-ChNF) were prepared from squid pens in this study. Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy proved that β-ChNFs with the diameter of 5 to 10 nm were prepared. β-ChNF dispersion became gelled upon the addition of cell culture medium. Cell culture experiments showed that β-ChNFs exhibited negligible cytotoxicity towards ADSCs and L929 cells, and it was found that more exosomes were secreted by the globular ADSCs grown in the β-ChNF hydrogel. The vivo experiments of rats showed that the ADSCs-loaded β-ChNF hydrogel could directly cover the wound surface and significantly accelerate the wound healing and promote the generation of epithelization, granulation tissue and collagen. In addition, the ADSCs-loaded β-ChNF hydrogel clearly regulated the expressions of VEGFR, α-SMA, collagen I and collagen III. Finally, we showed that ADSCs-loaded β-ChNF hydrogel activated the TGFβ/smad signaling. The neutralization of TGFβ markedly reduced Smad phosphorylation and the expressions of TIMP1, VEGFR and α-SMA. Taken together, these findings suggest that ADSCs-loaded β-ChNF hydrogel promises for treating wounds that are challenge to heal via conventional methods.