As　a　vital　enzyme　in　DNA　phosphorylation　and　restoration,　T4　polynucleotide　kinase　(T4　PNK)　has　aroused　great　interest　in　recent　years.　Therefore,　numerous　strategies　have　been　established　for　highly　sensitive　detection　of　T4　PNK　based　on　diverse　signal　amplification　techniques.　However,　they　often　need　sophisticated　design,　a　variety　of　auxiliary　reagents　and　enzymes,　or　cumbersome　manipulations.　We　have　designed　a　new　kind　of　allosteric　aptamer　probe　(AAP)　consisting　of　streptavidin　(SA)　aptamer　and　the　complementary　DNA　(cDNA)　for　simple　detection　of　T4　PNK　without　signal　amplification　and　with　minimized　interference　in　complex　biological　samples.　When　the　5′-terminus　of　the　cDNA　is　phosphorylated　by　T4　PNK,　the　cDNA　is　degraded　by　lambda　exonuclease　to　release　the　fluorescein　amidite　(FAM)-labeled　SA　aptamer,　which　subsequently　binds　to　streptavidin　beads.　The　enhancement　of　the　fluorescence　signal　on　SA　beads　can　be　detected　precisely　and　easily　by　a　microscope　or　flow　cytometer.　Our　method　performs　well　in　complex　biological　samples　as　a　result　of　the　enrichment　of　the　signaling　molecules　on　beads,　as　well　as　simple　manipulations　to　discard　the　background　interference　and　nonbinding　molecules.　Without　signal　amplification　techniques,　our　AAP　method　not　only　avoids　complicated　manipulations　but　also　decreases　the　time　required.　With　the　advantages　of　ease　of　operation,　reliability,　and　robustness　for　T4　PNK　detection　in　buffer　as　well　as　real　biological　samples,　the　AAP　has　great　potential　for　clinical　diagnostics,　inhibitor　screening,　and　drug　discovery.　©　2017　American　Chemical　Society.