Tetracyclines are a group of important antibiotics with a common four-ring scaffold. While most tetracyclines are currently used only in animals, their leaching into the environment and residues in food have caused health concerns. Aptamers are an attractive way to detect tetracyclines, and all previously reported aptamers for tetracyclines were obtained by immobilizing target molecules. In this work, we selected a few DNA aptamers by immobilizing the DNA library using oxytetracycline as the target. We obtained new aptamers with no overlapping sequences compared to the previously reported ones, and a representative sequence named OTC5 had a dissociation constant of 147 nM measured by isothermal titration calorimetry. Similar binding affinities were also observed with tetracycline and doxycycline. Because tetracyclines are fluorescent and their fluorescence intensity was enhanced by binding to the aptamers, a label-free and dye-free fluorescent biosensor was developed with a detection limit of 25 nM oxytetracycline. The sensor was able to detect targets in milk after extraction. Fluorescence polarization measurement showed that this aptamer is insensitive to sodium concentration but requires magnesium. Finally, a strand-displacement biosensor was designed, and it has a detection limit of 1.2 μM oxytetracycline.