The hydrogel nanoparticles can be prepared using linoleic modified chitosan following sonication. The morphology and size distribution of nanoparticles were determined by AFM and photocorrelation spectroscopy, respectively. The effects of environment factors on particle size and adsorption of trypsin (TR) were also analyzed. The LA-chitosan nanoparticles were in the range of 200–600 nm with satisfactory structural integrity. The particle size slightly increased with an increase in concentration. In high ionic strength solution the particle size decreased, compared to salt-free solution, on the other hand, the particles size increases as urea concentration increased. The particle size was found to be larger in acidic solution than in neutral and alkaline solution. Environment factors (e.g. pH, concentration of urea or NaCl) can affect TR loading on the nanoparticle. The thermal stability of TR loading on nanoparticles was significantly improved compared to free TR.