This study developed a thymine-based molecular beacon (MB), which can be used for screening triplex DNA binders and quantifying coralyne and sanguinarine. Triplex DNA binder triggered the formation of triple-stranded DNA between MB and 7-repeat adenosine (A₇), switching off the fluorescence of MB. Based on the use of triplex DNA binder and A₇ as inputs and the fluorescence change of MB as the output, the NAND gate was constructed to discriminate triplex DNA binders from duplex DNA binders.