With the fast changing lifestyle, vitamin D deficiency is becoming extremely common. Therefore, development of economical, efficient, and fast sensors for vitamin D is the need of the hour. Carbon-based nanomaterials are extensively explored in sensing of variety of biomolecules. In the present study, an antibody-free, highly sensitive, carbon-nanotube-based, highly responsive vitamin D3 sensor is reported. Nitrogen-doped carbon nanotubes are utilized to overcome the limiting factor of hydrophobic character of pure carbon. The synthesized N-doped CNTs showed a specific surface area of 24 m 2 /g. The surface charges of vitamin D3 and the vitamin D3/NCNT complex are found to be −20 and −6.4 mV, respectively, by zeta potential measurements. The sensor is able to deliver high performance in the concentration range of 0−10 nM, with a limit of detection of 16 pM. The response study indicated the sensitivity value as 0.000495 mA/cm 2 nM. The sensor is also able to show a higher selectivity toward vitamin D3 in comparison to other biomolecules. The long-term stability, reproducibility, good linear range, and ultralow detection capability of the sensor are also reported.