Vitamin D is an essential metabolite, and its deficiency causes many skeletal and chronic diseases. 25-hydroxyvitamin D₃ (25(OH)D₃) is known as a vitamin D biomarker and is widely used in clinical diagnosis to determine vitamin D status in human bodies. Herein, we propose a novel electrochemical aptasensor for the sensitive detection of 25(OH)D₃ via the employment of DNA tetrahedron and catalytic hairpin assembly (CHA) amplification strategy. The electrochemical aptasensor was fabricated by the immobilization of the DNA tetrahedron on the Au surface, followed by assembling the hairpin DNA probe-25(OH)D₃ complex (HP1-D). The HP1-D could be replaced by the HP2 hairpin and released for initiating CHA cycles. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) had been employed to study the electrochemical properties of the developed sensor. The designed electrochemical aptasensor showed a wide linear range of 0.1–1000 nM with a low detection limit (LOD) of 0.026 nM for the detection 25(OH)D₃ with high selectivity, stability, and reproducibility. Furthermore, the proposed sensor was demonstrated in human serum to detect 25(OH)D₃, indicating that it has a promising potential application in the clinical diagnosis of vitamin D deficiency.