Polynucleotide　kinase　(PNK)　is　an　important　DNA　damage　repair-related　enzyme　and　also　a　promising　therapeutic　target　in　various　diseases.　Here,　we　developed　a　dual　amplified　sensing　strategy　based　on　the　combination　of　terminal　deoxynucleotidyl　transferase　(TdT)　and　CRISPR-Cas12a　for　high　selective　and　sensitive　detection　of　PNK　activity　in　cell　lysates　and　also　inhibitor　screening.　In　this　sensing　system,　the　PNK　converted　the　3＇-phosphate　DNA　to　3＇-hydroxy　DNA,　and　then　the　TdT　catalyzed　to　add　a　long　poly-adenine　(poly　(A))　at　the　3＇　OH　terminus　DNA　(as　first　amplified　step).　The　produced　poly　(A)　tail　acted　as　activator　to　trigger　the　Cas12a　trans-cleavage　(non-target)　activity　to　cleave　reporter　probes　for　fluorescence　detection　(as　second　amplified　step).　The　dual　amplified　steps　lead　to　a　synergetic　signal　amplification　effect　for　sensitive　detection　of　PNK　activity.　The　developed　sensing　platform　enable　detect　PNK　activity　with　linear　range　from　0　to　1　U/mL　and　a　direct　detection　limit　of　5　x　10(-4)　U/mL.　It　was　also　successfully　used　for　PNK　inhibitor　screening　and　PNK　activity　detection　in　cell　extracts　even　at　a　single-cell　level.　The　efficient　dual　amplified　method　can　broaden　the　CRISPR/Cas-based　sensing　systems　in　other　bio-/chem-analysis　fields.