Human epidermal growth factor receptor-2 (HER2) has been recognized as an important biomarker for the early diagnosis and management of breast cancer. However, there is still challenge in the clinical detection of HER2. In this work, a simple strategy for "turn on" fluorescent detection of HER2 with ultra-sensitivity and high specificity was developed. Herein, HER2-binding aptamer (HApt) and DNA2 containing G-rich sequences, templated sequences for Ag nanoclusters (AgNCs), and complementary bases at both ends were involved to achieve the double stranded DNA templated AgNCs (dsDNA-AgNCs) as a fluorescence probe for HER2 detection. In the presence of HER2, the highly specific binding of HER2 to HApt caused HApt separating from dsDNA-AgNCs, resulting in the folding of DNA2-AgNCs because of the complementary bases at both ends, which led to AgNCs' proximity to G-rich sequences, and therefore the enhanced fluorescence intensity. By monitoring the change in fluorescence signal (ΔF), HER2 was measured, and a linear range from 8.5 fM to 225 fM with a limit of detection (LOD) 0.0904 fM was obtained. More significantly, the detection of HER2 in serum samples was achieved with high accuracy, and the breast cancer patients were successfully discriminated from healthy persons. In summary, this strategy is simple, time-saving, cost-effective, ultrasensitive, specific, universal and more applicable for the detection of HER2. Therefore, we expect this present aptasensor can be used in the clinical detection of biomakers, which lays a potential foundation for the early diagnosis of cancer.