The aim of this study was to develop an effective wound dressing using a temperature-responsive hydroxybutyl chitosan (HBC) based hydrogel. The HBC - chitosan (CS) - dopamine (HCS-DOPA) composite hydrogels were prepared by the dopamine self-polymerization at different concentrations (0, 0.5, 1.0 and 2.0 mg/mL), termed as HCS, HCS-DOPA-0.5, HCS-DOPA-1 and HCS-DOPA-2, respectively. The gelling characteristic of HBC hydrogel was not influenced by composite CS and DOPA. The HCS-DOPA composite hydrogels were non-cytotoxic to mouse fibroblast cells (L929), and induced under 5.0% hemolysis rate. In vitro antibacterial studies, composite HCS-DOPA-2 hydrogels exhibited lasting inhibition to S. aureus >8 h. The whole blood test in vitro demonstrated that blood clotting time treated with HCS-DOPA-2 composite hydrogels was shortened to 95.6 s compared with that of HCS in vitro hemostasis. The results suggested that HCS-DOPA-2 composite hydrogels could be applied as a promising wound dressing for hemostasis in vitro. •The HCS-DOPA hydrogels emerged a special porous structure with uniform pore size and had phase transition between gel-sol.•The composite HCS-DOPA hydrogels were non-cytotoxic to mouse fibroblast cells (L929), and induced under 5.0% hemolysis rate.•The composite HCS-DOPA hydrogels exhibited lasting inhibition of S. aureus more than 8 h in vitro antibacterial studies.•The composite HCS-DOPA hydrogels significantly shortened blood clotting time compared with that of HCS in vitro hemostasis.