We describe the characterization of a DNA aptamer that displays high affinity and specificity for the anthracyclines daunomycin and doxorubicin, both of which are frequently used in chemotherapy. Aptamers were isolated from a pool of random sequences using a semiautomated procedure for magnetic beads. All selected aptamers displayed high affinity for the target molecule daunomycin. One aptamer was further characterized and exhibited a dissociation constant (KD) of 20 nM. To examine the aptamer's binding properties and clarify its applicability for diagnostic assays, its performance under various buffer conditions was evaluated. The aptamer proved to be very robust and not dependent on the presence of specific ions. It also tolerated a wide pH range and immobilization via 5'-biotinylation. Furthermore, a competition assay for sensitive daunomycin detection was established. This not only allows the determination of the aptamer's specificity but also allows the quantification of as little as 8.4 microg/L daunomycin and doxorubicin.