Vitamin D deficiency remains a serious health issue in many developing countries and regions with a deficiency of sunlight. And although the demand for vitamin D testing has increased dramatically in recent years, finding new methods for efficient rapid testing remain at the forefront for clinical diagnosis and public health prevention. Herein, we are introducing an electrochemical aptasensor for the detection of 25-hydroxyvitamin D₃ (25(OH)D₃), comparing two aptamers platforms. The oligonucleotide aptamers were immobilized on gold surfaces using a disulfide linker, followed by monitoring the electrochemical response by cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Our studies revealed that the aptamer VDBA14 possessing a 5′-terminal disulfide recognizes the vitamin target. This electrochemical aptasensor exhibited a wide linear range of 1–1000 nM and a limit of detection (LOD) of 0.085 nM for the detection of 25(OH)D₃. The aptasensor also allows the detection of 25(OH)D₃ in human serum sample with high sensitivity and selectivity, indicating that the proposed electrochemical aptasensor may have potential applications in clinical diagnosis.