Nisin is a high-efficiency Gram-positive bacterium-targeted antimicrobial peptide widely used in the food industry. However, its antibacterial activity can be attenuated in specific niches, and its biomedical applications are limited. Herein, nisin was covalently bonded with gellan gum, a biocompatible polysaccharide. The antibacterial stability of nisin was also verified to be effectively enhanced. After nisin was conjugated with gellan, the antibacterial duration of nisin against Staphylococcus epidermidis increased from 96 h to 216 h under acidic conditions and from 48 h to 144 h under alkaline conditions. The antibacterial activity of gellan-nisin conjugate was also not lost with either heat alkali treatment at 80 °C or chymotrypsin digestion. Hemolysis and cytotoxicity tests also confirmed the good biocompatibility of this conjugate. This conjugate could be a promising biomaterial for medical applications, such as wound dressings and implant coatings.