Gelatin nanoparticles, cross-linked by a mixture of a water soluble carbodiimide (CDI) and N-hydroxysuccinimide (NHS) as a non-toxic cross-linking system, was prepared. The conventional two step desolvation method with acetone as the non-solvent was used. The mean size and size distribution as well as the morphology of the formed nanoparticles were evaluated and compared with those of nanoparticles cross-linked by glutaraldehyde (GA) as the most commonly used cross-linking agent. Furthermore, intrinsic viscosities of the nanoparticles cross-linked by CDI/NHS and GA were measured and compared under various conditions. The results showed the formation of smoother and more homogeneous nanoparticles with smaller size when CDI/NHS used as cross-linking agent under the same synthesis condition. Moreover, nanoparticles encapsulating paracetamol as a model drug were produced by the two different cross-linking agents and were characterized for drug entrapment and loading efficiencies and in vitro drug release. Both drug entrapment and loading efficiencies was higher in the CDI/NHS cross-linked nanoparticles; however, the release kinetics was comparable to that of nanoparticles cross-linked with GA. The differences in the characteristics of CDI/NHS and GA cross-linked nanoparticles were attributed to the different nature of network structures formed by the two cross-linking agents. On the whole, these results suggested that CDI/NHS cross-linked nanoparticles have high potential to be used for drug delivery application in preference to the nanoparticles synthesized by toxic cross-linking agents.