Immunoglobulin　new　antigen　receptor　(IgNAR)　is　a　naturally　occurring　antibody　that　consists　of　only　two　heavy　chains　with　two　independent　variable　domains.　The　variable　binding　domain　of　IgNAR,　called　variable　new　antigen　receptor　(VNAR),　is　attractive　due　to　its　solubility,　thermal　stability,　and　small　size.　Hepatitis　B　surface　antigen　(HBsAg)　is　a　viral　capsid　protein　found　on　the　surface　of　the　Hepatitis　B　virus　(HBV).　It　appears　in　the　blood　of　an　individual　infected　with　HBV　and　is　widely　used　as　a　diagnostic　marker　for　HBV　infection.　In　this　study,　the　whitespotted　bamboo　sharks　(Chiloscyllium　plagiosum)　were　immunized　with　the　recombinant　HBsAg　protein.　Peripheral　blood　leukocytes　(PBLs)　of　immunized　bamboo　sharks　were　further　isolated　and　used　to　construct　a　VNAR-targeted　HBsAg　phage　display　library.　The　20　specific　VNARs　against　HBsAg　were　then　isolated　by　bio-panning　and　phage　ELISA.　The　50%　of　maximal　effect　(EC50)　of　three　nanobodies,　including　HB14,　HB17,　and　HB18,　were　4.864　nM,　4.260　nM,　and　8.979　nM,　respectively.　The　Sandwich　ELISA　assay　further　showed　that　these　three　nanobodies　interacted　with　different　epitopes　of　HBsAg　protein.　When　taken　together,　our　results　provide　a　new　possibility　for　the　application　of　VNAR　in　HBV　diagnosis　and　also　demonstrate　the　feasibility　of　using　VNAR　for　medical　testing.　©　2023　by　the　authors.