The aim of this research was to investigate the potential of ampicillin-loaded hydrogels based on polyvinyl alcohol (PVA), hyaluronic acid (HA) and kappa-carrageenan ((K)-Carr) as an antibiotic-releasing wound dressing. Firstly, the novel polymers (HA-g-(K)-Carr) were synthesized by grafting of HA onto (K)-Carr using 4-dimethylaminopyridine/1-(3-dimethylaminopyl)-3-ethyl-carbodiimide hydrochloride as catalyst system via esterification reaction. The characterization of the polymer structure was performed by Fourier transform infrared spectrum (FTIR), proton nuclear magnetic resonance and thermogravimetric analysis. Secondly, PVA/(HA-g-(K)-Carr) hydrogel with/without loading of ampicillin molecules was formed via freeze-thawing method since PVA/K-Carr and PVA/HA hydrogels were also fabricated as control groups. The hydrogels were subjected to characterizations with FTIR and X-ray diffractometer. PVA/(HA-g-(K)-Carr) hydrogel demonstrated the highest swelling amount and highest ampicillin release amount reaching an equilibrium value after 480 min rather than the other hydrogels. Also, PVA/(HA-g-(K)-Carr) hydrogel exhibited inhibition zone against Escherichia coli and Staphylococcus aureus and no cytotoxic effect for L929 cells. All the results showed that PVA/(HA-g-(K)-Carr) hydrogels are good candidates for wound dressing applications.