CD44 is a promising biomarker in the diagnosis and prognosis of malignancies. The serum CD44 level is closely related to disease progression and metastasis of malignancies. It is of great clinical significance for the detection of serum soluble CD44. In this study, a facile, label-free aptamer based electrochemical impedance sensor for serum CD44 has been proposed. The aptamer showing high affinity to CD44 was immobilized on the gold electrodes through Au-S interaction. The interaction between target CD44 and the immobilized aptamer will cause a complex structure change of the aptamer, which makes the diffusion of [Fe(CN)6]3-/4- toward the electrode surface easy, thus resulting in the decrease of the impedance of the system. The decreased degree of the impedance had a good linear relationship with the logarithm of the CD44 concentration in the range of 0.1-1000 ng mL-1 with a detection limit of 0.087 ng mL-1 (S/N = 3). The developed biosensor has been applied to detect CD44 in serum samples with satisfactory results.