A　chloramphenicol　(CAP)-binding　aptamer　of　80　nucleotides　(nt)　was　reported　in　2011.　In　2014,　it　was　truncated　to　40　nt　and　has　since　been　used　by　most　researchers,　although　a　careful　binding　study　is　still　lacking.　In　this　work,　binding　assays　using　isothermal　titration　calorimetry　and　various　DNA-staining　dyes　were　performed.　By　comparing　the　truncated　aptamer　with　three　control　sequences,　no　specific　binding　of　CAP　was　observed　in　each　case.　The　secondary　structures　of　the　original　and　truncated　aptamers　were　analyzed,　and　it　was　shown　that　the　likelihood　of　the　truncated　aptamer　to　retain　the　same　binding　mechanism　as　the　original　sequence　is　low.　We　further　examined　gold　nanoparticle　(AuNP)-based　label-free　colorimetric　assays.　By　quantifying　the　extinction　ratio　at　620　nm　over　that　at　520　nm,　a　similar　color　response　was　observed　regardless　of　the　sequence　of　DNA,　suggesting　the　color　change　mainly　reflected　other　events　such　as　the　adsorption　of　CAP　by　the　AuNPs,　instead　of　aptamer　binding　to　CAP.　Salt-induced　aggregation　experiments　suggested　direct　adsorption　of　CAP　on　AuNPs.　CAP　only　weakly　inhibited　DNA　adsorption　by　AuNPs　but　did　not　displace　pre-adsorbed　DNA.　Therefore,　CAP　adsorption　by　AuNPs　needs　to　be　considered　when　designing　related　sensors,　for　example,　by　using　non-aptamer　sequences　as　controls.　This　work　calls　for　careful　confirmation　of　aptamer　binding　and　control　experiments　for　designing　aptamer　and　AuNP-based　biosensors.