Gentamycin (GM) and salicylic acid (SA) loaded chitosan (CS) nanoparticles are prepared and used to inhibit the ototoxicity of GM. Nanoparticles are prepared via the cross-linking method by CS and sodium tripolyphosphate. The characteristics of nanoparticles such as size, zeta potential, shape, loading capacity, and in vitro release profiles are determined. The nanoparticles are spherical in shape with an average diameter of 40 nm. The entrapment efficient of GM and SA is (91.24±0.24)% and (80.75±0.15)% respectively, and loading capacity are (34.15±1.02)% and (38.35±0.48)%. The drug release shows good sustained-release effect and follows ambiexponent kinetic equation. It demonstrates that GM and SA loaded CS nanoparticles have promising potential effect on antagonism ototoxicity of GM, and the nephrotoxicity may be also decreased through the expected sustained-release characteristics of CS nanoparticles.