Aptamers　are　short　single-stranded　DNA　or　RNA,　which　can　be　selected　from　a　random　combinatorial　library　by　the　systematic　evolution　of　ligands　by　exponential　enrichment　(SELEX)　in　vitro.　In　order　to　obtain　novel　selective　light-up　ligands　for　berberine,　we　selected　DNA　aptamers　for　berberine　by　a　modified　affinity　chromatography-based　SELEX.　The　ssDNA　library　was　coupled　on　streptavidin-coated　agarose　beads　through　the　hybridization　with　a　biotin　labeled　complementary　oligonucleotide　capture　strand,　and　the　aptamer　sequences　were　released　from　agarose　beads　through　berberine　binding.　This　method　to　isolate　the　aptamers　was　simple,　straightforward　and　effective,　and　three　aptamers　with　low　micromolar　dissociation　constants　(K(d)s)　were　achieved　after　only　6　rounds　of　selection.　The　best　of　them　was　further　optimized,　and　the　shortened　aptamers　showed　similar　K(d)s　to　the　full　length　sequences.　The　light-up　phenomenon　of　the　berberine-aptamer　pair　was　also　investigated　and　the　final　minimized　21-mer　aptamer　which　showed　higher　fluorescence　enhancement　than　G-quadruplex　structures　could　be　used　as　a　novel　light-up　fluorescent　probe　for　various　biosensing　applications.