Molecular　tags　such　as　fluorophores　are　increasingly　being　replaced　with　nanoparticles　thanks　to　their　superior　optical　properties,　substantial　chemical　stability,　and　stability　against　photobleaching.　Herein,　we　innovatively　constructed　a　new　ratiometric　fluorescence　enzyme-linked　immunosorbent　assay　(RF-ELISA)　for　the　screening　of　alpha-fetoprotein　(AFP)　in　early　hepatocellular　carcinoma　in　vitro　diagnostics　using　carbon　dots@SiO2@CdTe　quantum　dots　(CDs@SiO2@CdTe　QDs).　Carbon　dots　with　blue　fluorescence　were　initially　encapsulated　into　SiO2　nanospheres　through　the　typical　Stöber　method.　Thereafter,　CdTe　QDs　with　red　fluorescence　were　modified　onto　the　surface　of　CDs@SiO2　nanospheres.　Dual-emission　nanotags　with　blue　and　red　fluorescent　signals　were　utilized　to　design　a　RF-ELISA　method　for　the　determination　of　AFP　on　the　anti-AFP　capture　antibody-coated　microplate　using　glucose　oxidase　(GOx)-labeled　anti-AFP　secondary　antibody.　After　the　formation　of　the　sandwiched　immunocomplex,　GOx　catalyzed　glucose　to　generate　hydrogen　peroxide　(H2O2),　which　could　quench　the　red　fluorescence　of　CdTe　QDs　on　the　surface　of　nanotags.　Meanwhile,　the　encapsulated　carbon　dots　in　the　nanotags　could　still　maintain　the　initial　blue　fluorescence　intensity.　The　ratio　between　red　fluorescence　intensity　and　blue-emission　intensity　could　be　used　for　the　quantitative　monitoring　of　AFP　concentration　under　optimum　conditions.　The　experimental　results　indicated　that　CDs@SiO2@CdTe　QDs-based　RF-ELISA　could　exhibit　a　good　fluorescence　signal　with　a　dynamic　linear　range　of　0.05-60　ng　mL−1　at　a　low　detection　limit　of　8.7　pg　mL−1.　Moreover,　the　fluorescence　color　of　the　solution　including　CDs@SiO2@CdTe　QDs　changed　from　pink　to　purple　to　blue　with　the　increasing　AFP　level　when　viewed　by　the　naked　eye.　Good　reproducibility,　high　specificity,　and　acceptable　stability　were　achieved　for　the　analysis　of　target　AFP.　Importantly,　the　accuracy　of　ratiometric　fluorescence　immunoassay　was　evaluated　to　determine　human　serum　samples,　giving　well-matched　results　relative　to　commercially　usable　human　AFP　ELISA　method.　©　2022　The　Royal　Society　of　Chemistry