Enzyme-linked　immunosorbent　assay　(ELISA)　is　an　economic　and　easy　operation　technique　that　has　been　widely　used　for　the　detection　of　protein　in　industry.　However,　the　low　loading　capacity　of　the　enzyme　reporter　has　contributed　to　the　low　sensitivity　of　traditional　ELISA,　and　the　cross-linking　procedures　of　the　enzyme-labeled　antibody　in　ELISA　methods　can　lead　to　the　inactivation　of　the　enzyme,　which　will　further　decrease　the　sensitivity.　To　address　this　issue,　herein　we　fabricated　＇carrier-free＇nanoparticles　to　obtain　a　horseradish　peroxidase　(HRP)　labelled　reporter　with　a　high　HRP　loading　capacity.　A　disulphide-containing　bis-N-hydroxy　succinimide　(NHS)　crosslinker　(NHS-SS-NHS)　was　used　to　control　the　link　and　release　of　traceless　HRPs,　thus　without　reduction　of　its　enzymatic　activity.　The　HRP　nanoparticle　(NanoHRP)　was　successfully　applied　for　dot　blotting　and　ELISA.　When　carcinoembryonic　antigen　(CEA)　was　used　as　a　target,　the　detection　limit　of　the　NanoHRP-based　ELISA　was　0.005　ng　mL-1,　which　was　about　400　times　more　sensitive　than　traditional　ELISA.　A　good　correlation　between　the　CEA　concentrations　and　the　response　values　measured　by　NanoHRP　ELISA　was　obtained　in　the　range　of　0.005　to　1　ng　mL-1.　This　concept　could　be　exploited　to　improve　ELISA　tests,　especially　those　requiring　a　high　accuracy,　to　facilitate　physicians　in　deciding　the　appropriate　medical　treatment.　This　journal　is　©　The　Royal　Society　of　Chemistry.