A label-free electrochemical impedance biosensor was fabricated for adenosine detection based on a new combining mode of aptamer probes（AP） and hairpin probes（HP） in the presence of adenosine（AD）.AP was first modified on the surface of the electrode.In the presence of AD，AP was folded into hairpin structure，the loop of the hairpin hybrids with complementary loop of HP to form a“kissing aptamer complex”，through the loop-loop interactions（being similar to“kissing” behavior of the human）.Due to the steric effect of the complex，the impedance signal of the electrode was obviously increased.On the contrary，in the absence of adenosine，AP was unfolded，which was unable to combine with HP into a stable“kissing”structure.The impedance signal did not change significantly.In this study，the electron transfer resistance values before and after combination were used for the detection of adenosine.Under the optimized conditions，a good linear relationship was obtained between resistance value and logarithm of concentration of AD in the range of 5-1 000 nmol/L.The limit of detection for adenosine is as low as 0.6 nmol/L.In addition，the sensor has a good selectivity.Guanosine，cytidine and uridine did not influence the detection of adenosine.The sensor is expected to be applied in the detection of adenosine in clinical real samples.