In　most　aptamer　based　stimulus　response　mesoporous　silica　nanoparticles　(MSN)　systems,　the　aptamer　is　modified　on　the　MSN　via　electrostatic　interaction,　however　leakage　might　exist　after　a　certain　time　in　the　system　and　hence　the　stability　is　not　good.　In　this　study,　the　pores　of　MSN　were　capped　by　aptamer　through　click　chemistry　reaction　for　the　first　time　and　the　system　was　then　employed　to　develop　a　fluorescence　biosensor.　Specifically,　the　aptamer　of　the　target　(thrombin　in　this　study)　was　hybridized　with　its　complementary　DNA　(which　was　initially　modified　with　alkyne　at　the　terminal)　to　form　a　double　strand　DNA　(dsDNA)　firstly,　and　then　this　dsDNA　was　modified　on　N-3　modified　MSN　via　Cu(I)　catalyzed　alkyne-azide　cycloaddition　reaction.　The　guest　molecules　(fluorescein)　were　blocked　in　the　pores　of　the　MSN　with　high　efficiency　and　nearly　no　leakage　was　detected.　Upon　the　introduction　of　thrombin,　thrombin　specifically　recognized　its　aptamer,　so　aptamer　released　from　the　MSN;　and　the　single　strand　DNA(ssDNA)　left　could　not　cap　the　pores　of　the　MSN　efficiently　and　hence　caused　the　releasing　of　fluorescein　into　the　solution.　The　enhanced　fluorescence　intensity　of　the　system　has　a　good　linear　relationship　with　the　thrombin　concentration　in　the　range　of　50-1000　ng　mL(-1)　with　a　detection　limit　of　28.46　ng　mL(-1).　The　proposed　biosensor　has　been　successfully　applied　to　detect　thrombin　in　serum　samples　with　high　selectivity.　The　same　strategy　can　be　applied　to　develop　biosensors　for　different　targets　by　changing　the　adopted　aptamer.