Herein, we developed a sensitive sensing platform for tetracycline detection by combining the specific DNA aptamers with a novel TiO2 nanocomposite. In this composite, cylinder-shaped TiO2 nanorods were synthesized on MoS2 nanospheres by a facile hydrothermal method to produce a MoS2-TiO2 composite, which was then aminated. Following that, gold nanoparticles (Au) were deposited on the aminated MoS2-TiO2 to form MoS2-TiO2@Au composite, on which thiolated DNA aptamers were applied to construct an electrochemical aptasensor. The tetracycline aptamer could well hybridize to its complementary biotin-DNA oligonucleotide (bio-cDNA), and then avidin-horseradish peroxidase (avidin-HRP) mediated by H2O2 was introduced to produce amplified electrochemical response. The competition between tetracycline and bio-cDNA for binding of the aptamer would decrease the electrochemical signal, which was used for quantitative detection of tetracycline. The prepared aptasensor showed a wide linear range from 1.5 × 10−10 to 6.0 × 10−6 M, and a low detection limit of 5 × 10−11 M.