DNA　nanotechnology　can　be　used　to　precisely　construct　nanostructures　of　different　shapes,　sizes　and　surface　chemistry,　which　is　appreciated　in　a　variety　of　areas　such　as　biomaterials,　nanodevices,　disease　diagnosis,　imaging,　and　drug　delivery.　Enzymatic　degradation　resistance　and　cell-targeting　capability　are　indispensable　for　the　applications　of　DNA　nanostructures　in　biological　and　biomedical　fields,　and　is　challenging　to　rationally　design　the　desirable　nanoscale　DNA　materials　suitable　for　the　clinical　translation　by　the　existing　assembly　methodologies.　Herein,　we　present　a　simple　and　efficient　method　for　the　hierarchical　assembly　of　a　three-level　DNA　ring-based　nanostructure　(DNA　h-Nanoring)　in　a　precise　order,　where　DNA　compositions　at　the　primary　level,　the　second　level　and　the　third　level　are　a　single　DNA　ring,　two-ring-hybridized　duplex　and　uniform　complex　macro-cycle,　respectively.　Most　as-assembled　DNA　h-Nanorings　exhibit　the　regular　two-dimensional　cycle-shaped　structure　characterized　by　atomic　force　microscopy　(AFM).　The　Nanoring　exhibits　a　significantly　enhanced　resistance　to　enzymatic　attack,　such　that　it　can　remain　intact　in　10%　fetal　bovine　serum　(FBS)　for　24　h,　and　even　stably　exist　in　the　presence　of　nuclease　at　a　high　concentration.　More　importantly,　it　is　very　easy　to　modify　the　DNA　h-Nanoring　with　functional　moieties　(e.g.,　targeting　ligand　aptamer)　because　there　are　many　single-stranded　fragments　available　for　further　hybridization.　By　combining　with　receptor-targeted　Sgc8,　the　nanoring　can　be　used　to　accomplish　the　cell　imaging　and　criminate　target　CEM　cells　from　control　cells,　demonstrating　a　potential　platform　for　in　vivo　tumor　imaging　and　targeted　chemotherapeutics　delivery.