This study aimed to compare type A (GA) and type B gelatin (GB) nanoparticles in terms of physico-chemical properties and the release behavior of different model compounds (methylene blue, eosin, and sericin) incorporated in both nanoparticles. The GA and GB nanoparticles (397–501 nm in diameter) were successfully fabricated by water in oil emulsion technique following by the glutaraldehyde crosslinking. GB nanoparticles showed the higher crosslinking degree, consequently resulted in the slower degradation rate than the GA nanoparticles. The model compounds having different charge characteristics including the positive-charged methylene blue and the negative-charged eosin, were loaded in the nanoparticles. We found that the entrapment and loading efficiencies of methylene blue and eosin depended upon the type of gelatin nanoparticles. The positive-charged methylene blue could entrap in the negative-charged GB nanoparticles at the high entrapment and loading efficiencies (92% and 46%, respectively), possibly due to their attractive electrostatic interaction. Correspondingly, the negative-charged eosin would repel the GB nanoparticles which had the same charge, resulted in the low entrapment and loading efficiencies (25% and 12%, respectively). On the other hand, the entrapment and loading efficiencies of sericin, a model of active compound, in the nanoparticle could not be explained by the electrostatic interaction. The negative-charged GB nanoparticles could entrap the negative-charged sericin at the high extent. The different profiles of methylene blue, eosin and sericin released from the GA and GB nanoparticles were obtained. The release profiles of methylene blue and eosin were mainly influenced by the type of gelatin nanoparticles. The sericin-encapsulated nanoparticles were cultured with L929 cells in order to confirm the biological activities of sericin. The sericin-encapsulated nanoparticles and the control sericin at all sericin concentrations were not toxic to L929 cells. Interestingly, the cells cultured in the presence of GA and GB nanoparticles loaded with 8 mg/mL sericin produced the significant amount of collagen, compared to those cultured with pure sericin control. This was possibly due to that the sericin-encapsulated gelatin nanoparticles