Detection　of　specific　genes　related　to　drug　action　can　provide　scientific　guidance　for　personalized　medicine.　Taking　the　detection　of　a　single-nucleotide　polymorphism　(SNP)　genotyping　related　to　the　chronic　hepatitis　B　virus　(HBV)　therapy　as　an　example,　a　novel　biosensor　with　high　sensitivity　and　selectivity　was　developed　based　on　the　hyperbranched　rolling　circle　amplification　(HRCA)　in　this　work.　The　single-base　mutant　DNA　(mutDNA)　sequence　can　perfectly　hybridize　with　the　specially　designed　discrimination　padlock　probe　and　initiate　the　HRCA　reaction.　Subsequently,　a　great　abundant　of　double-strand　DNA　sequences　were　released　and　a　strong　fluorescence　signal　can　be　detected　after　adding　SYBR　Green　I.　In　particular,　the　enhanced　fluorescence　intensity　exhibits　a　linear　relationship　with　the　logarithm　of　mutDNA　concentration　ranging　from　0.1　nM　to　40　nM　with　a　low　detection　limit　of　0.05　nM.　However,　when　there　was　even　a　single　base　mismatch　in　the　target　DNA,　the　HRCA　was　suppressed　and　fluorescence　response　process　could　not　occur,　resulting　in　a　high　selectivity　of　this　biosensor.　Moreover,　this　detection　strategy　also　performs　well　in　human　serums,　demonstrating　its　potential　application　in　detecting　SNPs　in　real　biological　samples.