Recombinant human erythropoietin (rHuEPO) is one kind of important hematopoietic growth factor, which is widely used in anaemia treatment as well as sometimes abused by endurance athletes. Based on a set of anti-rHuEPO-α aptamers successfully in vitro isolated in our laboratory, we herein describe a novel magnetic beads-based aptameric real-time PCR assay for the accurate quantification of rHuEPO-α, which combined the specific recognition with amplification capability of aptamers. Two detection strategies, termed 'recognition-after-hybridization' and 'recognition-before-hybridization' respectively, were constructed and compared. Strategy B, i.e.'recognition-before-hybridization', was finally adopted as the preferred one to measure rHuEPO-α. A limit of detection (LOD) of 1 pmol/L rHuEPO-α and a wide dynamic range from 6 pmol/L to 100 nmol/L were obtained for physiological buffer. Furthermore, a LOD of 6 pmol/L was achieved for more complicated matrix-half diluted artificial urine. These results indicate that the anti-rHuEPO-α aptamer fits the high sensitive detection of rHuEPO-α very well. The use of the aptamer with magnetic beads-based real-time PCR allows a direct and novel assay for rHuEPO-α.