We propose an in situ and label-free method for detection of biomolecular recognition events by use of a nanochannel-ion channel hybrid device integrated with an electrochemical detector. The aptamer is first immobilized on the outer surface of the nanochannel-ion channel hybrid. Its binding with target thrombin in solution considerably regulates the mass-transfer behavior of the device owing to the varied surface charge density and effective channel size. Via the electrochemical detector, the changed mass-transport property can be monitored in real time, which enables in situ and label-free detection of thrombin-aptamer recognition. The solution pH has a significant influence on detection sensitivity. Under optimal pH conditions, a detection limit as low as 0.22 fM thrombin can be achieved, which is much lower than most reported work. The present nanofluidic device provides a simple, ultrasensitive, and label-free platform for monitoring biomolecular recognition events, which would hold great potential in exploring the functions and reaction mechanisms of biomolecules in living systems.