A　versatile　point-of-care　assay　platform　was　developed　for　simultaneous　detection　of　multiple　targets　based　on　a　microfluidic　paper-based　analytic　device　(μPAD)　using　a　target-responsive　hydrogel　to　mediate　fluidic　flow　and　signal　readout.　An　aptamer-cross-linked　hydrogel　was　used　as　a　target-responsive　flow　regulator　in　the　μPAD.　In　the　absence　of　a　target,　the　hydrogel　is　formed　in　the　flow　channel,　stopping　the　flow　in　the　μPAD　and　preventing　the　colored　indicator　from　traveling　to　the　final　observation　spot,　thus　yielding　a　signal　off　readout.　In　contrast,　in　the　presence　of　a　target,　no　hydrogel　is　formed　because　of　the　preferential　interaction　of　target　and　aptamer.　This　allows　free　fluidic　flow　in　the　μPAD,　carrying　the　indicator　to　the　observation　spot　and　producing　a　signal　on　readout.　The　device　is　inexpensive　to　fabricate,　easy　to　use,　and　disposable　after　detection.　Testing　results　can　be　obtained　within　6　min　by　the　naked　eye　via　a　simple　loading　operation　without　the　need　for　any　auxiliary　equipment.　Multiple　targets,　including　cocaine,　adenosine,　and　Pb2+,　can　be　detected　simultaneously,　even　in　complex　biological　matrices　such　as　urine.　The　reported　method　offers　simple,　low　cost,　rapid,　user-friendly,　point-of-care　testing,　which　will　be　useful　in　many　applications.　©　2015　American　Chemical　Society.