The subject invention provides materials and methods for single-step detection of target molecules in a sample. The methods and assays of the subject invention employ a dye-displacement strategy, in which aptamers complexed with a cyanine dye for sensitive and rapid detection of targets of interest. In the presence of a target, aptamer-target binding liberates the non-covalently bound aptamer-binding dye, resulting in optical changes that can be observed spectrophotometrically or with the naked eye. The methods and assays of the subject invention enable the colorimetric detection of targets of interest regardless of their structure, sequence, target-binding affinity, and physicochemical properties of their targets.