A short kanamycin-binding aptamer has been widely used for detecting kanamycin. One of the popular signaling methods is based on the color change of gold nanoparticles (AuNPs) to develop label-free colorimetric biosensors. The general perception was that aptamer binding to its target would inhibit aptamer adsorption by the AuNPs. This inhibited adsorption results in the aggregation of the AuNPs and a color change upon addition of salt. However, the potential adsorption of kanamycin was ignored. Herein, we carefully studied the adsorption of kanamycin on AuNPs and performed a comprehensive analysis using two mutated aptamers and a randomly sequenced DNA which were not supposed to bind kanamycin. In addition, a total of six antibiotics were studied over a wide concentration range. As low as 90 nM kanamycin can induce the aggregation of 3 nM citrate-capped AuNPs, indicating very strong adsorption of kanamycin. The color change was independent of DNA sequence, and all the tested sequences showed a similar color response, regardless of aptamer. Among the different antibiotics, kanamycin and streptomycin induced a color change but not the other four. Our results support an alternative mechanism that kanamycin and streptomycin adsorption by the AuNPs was the main reason for the color change instead of aptamer binding.