Background:　Enzyme-linked　immunosorbent　assay　(ELISA)　requires　the　simultaneous　maintenance　of　enzyme　activity　and　the　efficient　labelling　of　the　enzymes　on　the　antibodies.　Challenges　in　ELISA　include　the　enzyme　decomposition　under　harsh　conditions　and　the　requirement　for　secondary　antibodies　for　binding.　Preserving　enzyme　activity　and　simplifying　antibody　binding　is　a　crucial　prerequisite　for　expanding　the　applications　of　ELISA.　Most　encapsulation　cases　focus　on　enzyme　immobilization　in　MOFs/COFs,　while　the　synergetic　encapsulation　of　enzyme-biotin　in　DNA　nanoflowers　(DNFs)　and　their　labeling　of　capture　antibodies　to　construct　stable　streamlined　ELISA　platforms　was　few.　It　is　clear　that　a　novel　protocol　is　needed　that　is　facile,　capable　of　preserving　both　high　enzymatic　activity　and　efficient　antibody　binding　for　the　stable　ELISA.　Results:　We　proposed　a　novel　horseradish　peroxidase　(HRP)/biotin　co-encapsulated　DNA　nanoflowers　(HRP/biotin@DNFs)　for　the　synergistic　implementation　of　the　robust　enzyme　immobilization　using　rolling　circle　amplification　and　secondary　antibody-free　antibody　binding　using　biotin-streptavidin　ligation　in　ELISA.　Enzyme　immobilization,　abundant　antibody　binding　sites,　and　superior　protection　of　enzymes　were　achieved　in　synchrony.　High　enzyme　activity,　excellent　stability　and　chemical　tolerance　of　HRP/biotin@DNFs　were　obtained.　Especially,　HRP/biotin@DNFs　provides　95　%–85　%　and　91　%–81　%　of　its　original　activity　after　the　exposure　at　25　°C　and　37　°C　in　5　days,　respectively,　whereas　that　of　free　HRP　was　drastically　reduced　to　only　40　%　and　20　%　after　the　exposure　at　25　°C　and　37　°C　for　5　days,　respectively.　Using　tetrabromobisphenol　A　bis-(2-hydroxyethyl)　ether　(viz.　TBBPA-BHEE)　as　the　target　model,　high　performance　for　the　assay　was　achieved,　with　high　enzymatic　activity,　good　tolerance　and　sensitive　limit　of　detection　of　0.66　ng　mL−1　that　was　5-fold　more　sensitive　than　that　of　common　ELISA.　Stable　and　accurate　ELISA　for　TBBPA-BHEE　was　realized　with　recovery　yields　of　91.4　%–112.8　%　with　RSD　at　1.6–3.4　%.　Significance:　This　study　pioneers　a　simple　yet　powerful　strategy　that　simultaneously　achieves　both　affordable,　biologically　encapsulated　enzymes　with　high　activity　and　secondary　antibody-free　antibody　conjugation.　It　expands　the　toolbox　of　enzyme　immobilization　and　facilitates　enzyme　binding　on　antibody　without　the　need　of　secondary　antibody,　enabling　cost-effective　and　robust　ELISA　for　environmental　assays.　©　2025　Elsevier　B.V.