Nisin is an antibiotic peptide widely used in food and pharmaceutical industry. To improve its efficiency, nisin was encapsulated in a poly-γ-glutamic acid (γ-PGA) and chitosan nanoparticle system using self-assembly method. The effects of the nisin concentration and chitosan coating on the properties of the nanoparticles were investigated. The release behavior and transport mechanism of nisin from the nanoparticles were also examined under different conditions (temperature of 4 and 25 °C and pH of 3.0, 6.0 and 8.0 respectively). Results showed that the loading content (LC) and mean particle size of the nanoparticles were increased with the increasing of nisin concentration, but encapsulation efficiency (EE) was declined. The chitosan coating enhanced the stability of nanoparticle (higher ζ potential) and led to a higher EE and LC, and bigger mean particle size. FT-IR spectroscopy suggested that the formation of nisin/γ-PGA/chitosan nanoparticle (N–P–C-NPs) was mainly attributed to the interactions among the function groups of chitosan, γ-PGA and nisin by hydrogen bonding and electrostatic interactions. The release of nisin from the nanoparticles exhibited a pH-dependent pattern, and the experimental data were fitted to a linear superimposition model which suggested that the release mechanism of nisin from the nanoparticles was an anomalous behavior. In addition, the in vitro antibacterial test showed that N–P–C-NPs was more efficient than nisin/γ-PGA nanoparticle or nisin in inhibiting the growth of Escherichia coli O157:H7 and Listeria monocytogenes, suggesting N–P–C-NPs could be developed as a promising novel food preservative.