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Antigen delivery framework utilising polymer-based nanomaterial is broadly used in biomedicine because of their high efficacy, antigenicity and biocompatibility. In this study, hepatitis B surface antigen fabricated chitosan-polyethylene glycol nanocomposite (HBsAg-CS-PEG NC) was prepared by in situ ionic gelation method which brought about highly stable nanoformulation that was characterised by electron microscopy, atomic force microscopy (AFM) Fourier transform infrared spectroscopy (FTIR). Particles morphology by electron microscopy studies reveals spherical, electron-dense, nanostructure with 100-120 nm. AFM studies show a uniform distribution of particles with distinct roughness. Specific interaction of polymers with the vaccine or surface modification and functionalization were confirmed by FTIR analysis. Synthesised nanocomposite exhibited high antigen loading and entrapment efficiency. Release profile study carried out under in vitro condition indicates that a gradual increase in release at increasing time intervals which confirms noteworthy stability and release pattern. Enhanced immunogenicity of the nanocomposite was determined by induction of rosette formation and rate of phagocytosis using T cell rosette formation assay (TCRF assay) and mouse leukemic monocyte macrophage phagocytosis assay. Notable enhancement on rosette formation and phagocytosis percentage was recorded in nanocomposite treatment as dose-dependent manner. Biocompatibility or non-target effect was done by evaluating acute or subacute toxicity against Wistar rat model and hemocompatibility. Hemocompatibility was measured by determination of hemolysis, antioxidative enzymes, erythrocytes aggregation and serum protein profiling. No sign of toxicity and mortality was observed in all the tested parameters of nanocomposite treated animal model. Hemocompatibility studies indicate that nanocomposite treatment was not shown hemolysis, changes in antioxidative enzymes and serum protein profiling which all confirms the best biocompatibility of the synthesised nanocomposite. Phytotoxic effect of the nanocomposite studied with Vigna mungo, Vigna radiata) and Brassica nigra seeds indicate that the nanocomposite treatment was not affecting seedlings emergence or germination index. All these findings unmistakably uncover that the nanocomposite fabricated HBsAg vaccine nanoformulation utilised as a successful immunising agent which constructed from its high immunogenic property and best biocompatibility against appropriate test model framework and investigated another sight in inoculation immunology part., (Copyright © 2019 Elsevier B.V. All rights reserved.)