Convenient and rapid detection of alpha fetoprotein (AFP) is vital for early diagnosis of hepatocellular carcinoma. In this work, low-cost (0.22 USD for single sensor) and stable (during 6 days) electrochemical aptasensor was developed for highly sensitive and direct detection of AFP in human serum with the assist of vertically-ordered mesoporous silica films (VMSF). VMSF has silanol groups on the surface and regularly ordered nanopores, which could provide binding sites for further functionalization of recognition aptamer and also confer the sensor with excellent anti-biofouling capacity. The sensing mechanism relies on the target AFP-controlled diffusion of Fe(CN)63−/4−redox electrochemical probe through the nanochannels of VMSF. The resulting reduced electrochemical responses are related to the AFP concentration, allowing the linear determination of AFP with a wide dynamic linear range and a low limit of detection. Accuracy and potential of the developed aptasensor were also demonstrated in human serum by standard addition method.