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An electrochemical aptamer-based sensor was designed to detect tetracycline (Tet) in chicken ham. First, polyacrylonitrile (PAN) polymer was used to prepare electrospun PAN nanofibers. PAN nanofibers were heat treated to form electrospun carbon nanofiber (CNF) mat. The electrospun CNF mat electrode was modified with Au and the aptamer of Tet was placed on the mat electrode. Raman spectroscopy and scanning electron microscopy equipped with energy-dispersive spectroscopy were used to identify the structure and morphology of electrospun CNFs, respectively. The behavior of CNF mat electrode was evaluated via cyclic voltammetry. The designed aptamer biosensor had a good repeatability, reproducibility, stability, and specific function for the detection of Tet in chicken ham. This biosensor was able to detect the Tet in the linear range of 1 × 10–9 M to 1 × 10–4 M with the detection limit of 1.2 × 10–10 M and preserve about 95% of its primary response after 20 days. In addition, the recoveries of Tet were achieved ranging from 96.92 to 104.31%. [ABSTRACT FROM AUTHOR]