Recently, the versatility of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide conjugates, which have been developed from the DNA-binding antibiotics distamycin A and netropsin, has been shown. These synthetic small molecules can permeate cells to bind with duplex DNA in a sequence-specific manner, and hence can influence gene expression in vivo. Accordingly, several reports demonstrating the sequence specificity and biological activity of Py-Im polyamides have accumulated. However, the benefits of Py-Im polyamides, in particular those conjugated with fluorophores, has been overlooked. Moreover, clear directions for the employment of these attractive artificial small molecules have not yet been shown. Here, we present a detailed overview of the current and prospective applications of Py-Im polyamidefluorophore conjugates, including sequence-specific recognition with fluorescence emission properties, and their potential roles in biological imaging.