In this study, gelatin-oleic conjugate (GOC) was synthesized as a biomaterial for the preparation of anti cancer drug loaded nanoparticles (NP). Oleic acid (OA) was covalently bound to gelatin via EDC/NHS reaction in water-ethanol cosolvent to form GOC. Fourier Transform Infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance (1H-NMR) study indicated the successful synthesis of GOC. The percentage of gelatin amino groups reacted with OA was up to 50% as determined by 2,4,6-trinitrobenzene sulfonic acid (TNBS) method. The gelatin-oleic nanoparticles (GON) were then prepared by desolvation method using glutaraldehyde and genipin as crosslinkers. Irinotecan was used as a model drug and loaded in GON by incubation and in-process adding method for comparison. Dynamic light scattering (DLS) and Transmission Electron Microscopy (TEM) data showed that the sizes of GON and irinotecan loaded GON (IRT-GON) were below 250 nm which is suitable for passive tumor targeting. The zeta potentials of GON and irinotecan loaded GON (IRT-GON) were below -20 mV which is inferred to be stable in suspension against the aggregation process. The incubation method was more suitable for drug loading because it did not affect the formation process of GON and thus did not increase the size of GON much compared to in-process adding method. The lipophilic property of oleic moiety in GOC increased the affinity between GOC molecules, thus reducing the amount of crosslinking agents needed to stabilize GON compared to gelatin nanoparticle (GN). Therefore, the toxicity of GON can be reduced and the applicability may be improved. These results showed that GOC is a promising material to prepare drug encapsulated NP and IRT-GON is a potential delivery system for cancer therapy.