A novel strategy to improve the sensitivity of molecularly imprinted polymer sensors based on a double-layered molecularly imprinted polymer hybrid film-modified gold electrode was investigated for the determination of testosterone. A conductive poly(anilinomethyltriethoxysilane) film was electrodeposited on the surface of the electrode, and a molecularly imprinted polysiloxane thin film was formed on the conductive layer by a sol-gel process to enhance the electrochemical response. The performance of the sensor for testosterone was investigated by square-wave voltammetry in the presence of a hexacyanoferrate/ferrite as a redox probe. The prepared sensor provided high recognition for testosterone with a broad linear range from 10 to 100fM and a low detection limit of 10fM. Good sensitivity, selectivity, and stability were observed with relative standard deviations less than 5%. Good recovery of testosterone was obtained when the sensor was used for the determination of testosterone in fortified urine.