An　integrated　distance-based　origami　paper　analytical　device　(ID-oPAD)　is　developed　for　simple,　user　friendly　and　visual　detection　of　targets　of　interest.　The　platform　enables　complete　integration　of　target　recognition,　signal　amplification,　and　visual　signal　output　based　on　aptamer/invertase-functionalized　sepharose　beads,　cascaded　enzymatic　reactions,　and　a　3D　microfluidic　paper-based　analytical　device　with　distance-based　readout,　respectively.　The　invertase-DNA　conjugate　is　released　upon　target　addition,　after　which　it　permeates　through　the　cellulose　and　flows　down　into　the　bottom　detection　zone,　whereas　sepharose　beads　with　larger　size　are　excluded　and　stay　in　the　upper　zone.　Finally,　the　released　conjugate　initiates　cascaded　enzymatic　reactions　and　translates　the　target　signal　into　a　brown　bar　chart　reading.　By　simply　closing　the　device,　the　ID-oPAD　enables　a　sample-in-answer-out　assay　within　30　min　with　visual　and　quantitative　readout.　Importantly,　bound/free　probe　separation　is　achieved　by　taking　advantage　of　the　size　difference　between　sepharose　beads　and　cellulose　pores,　and　the　downstream　enzymatic　amplification　is　realized　based　on　the　compatibility　of　multiple　enzymes　with　corresponding　substrates.　Overall,　with　the　advantages　of　low-cost,　disposability,　simple　operation,　and　visual　quantitative　readout,　the　ID-oPAD　offers　an　ideal　platform　for　point-of-care　testing,　especially　in　resource-limited　areas.　©　2017　American　Chemical　Society.