A sensitive and efficient ratiometric electrochemical aptasensor was designed for tetracycline (TET) detection in milk. The ratiometric electrochemical aptasensor was constructed by integrating two aptasensors termed as aptasensor 1 and aptasensor 2. The aptasensor 1 was fabricated that based on ferrocene (Fc) and gold nanoparticles (AuNPs) nanocomposite. Meanwhile, the aptasensor 2 was prepared that based on carbon nanofibers (CNFs) and AuNPs nanocomposite. TET-aptamer was immobilized effectively onto screen-printed carbon electrodes (SPCEs) surface through forming Au-S bond between AuNPs and thiol of aptamer at 5' end to construct the aptasensor 1 and aptasensor 2. And their detection results were calculated by ratio. Thus, the proposed ratiometric aptasensor solved the problem of low accuracy and large differences between batches. Under the optimized conditions, the TET was detected by differential pulse voltammetry (DPV). Taken advantage of ratio calculation, the as-prepared ratiometric aptasensor could detect TET quantitatively in the range of 10-8-10-3gL-1, with a detection limit of 3.3 × 10-7gL-1. Moreover, its applicability to TET-contaminated real samples (milk) showed an excellent agreement with the values determined by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS). With high sensitivity, accuracy and reliability, the developed ratiometric aptasensor held a great potential in TET detection for food safety.