DNA　nanostructures　with　controllable　motions　and　functions　have　been　used　as　flexible　scaffolds　to　precisely　and　spatially　organize　molecular　reactions　at　the　nanoscale.　The　construction　of　dynamic　DNA　nanostructures　with　site-specifically　incorporated　functional　elements　is　a　critical　step　toward　building　nanomachines.　Artificial　self-assembled　DNA　nanostructures　have　also　been　developed　to　mimic　key　biological　processes　like　various　small　biomolecule-　and　protein-based　functional　biochemistry　pathways.　Here,　we　report　a　self-assembled　dynamic　trident-shaped　DNA　(TS　DNA)　nanoactuator,　in　which　biomolecules　can　be　tethered　to　the　three　＂arms＂　of　the　TS　DNA　nanoactuator.　The　TS　DNA　nanoactuator　is　implemented　as　the　mechanical　scaffold　for　the　reconfiguration　of　fluorescent/quenching　molecules　and　the　assembly　of　gold　nanoparticles,　which　exhibit　controlled　spatial　separation.　Furthermore,　two　enzymes　(glucose　oxidase　and　horseradish　peroxidase)　are　attached　to　the　two　outer　arms　of　the　TS　DNA　nanoactuator,　which　show　an　enhanced　cascade　reaction　efficiency　compared　to　free　enzymes.　The　efficiency　of　the　two-enzyme　cascade　reaction　can　be　spatially　regulated　by　switching　the　TS　DNA　nanoactuator　between　opened,　semiopened,　and　closed　states　through　adding　the　＂thermodynamic　drivers＂　(fuels　or　antifuels).　This　is　the　first　report　to　precisely　modulate　the　relative　position　of　coupled　enzyme　with　multiple　states　and　only　based　on　one　dynamic　DNA　scaffold.　The　present　TS　DNA　nanoactuator　with　multistage　conformational　transition　functionality　could　be　applied　as　a　potential　platform　to　precisely　and　dynamically　control　the　multienzyme　pathways　and　would　broaden　the　scope　of　DNA　nanostructures　in　single-molecule　biology　applications.　©　2018　American　Chemical　Society.