A voltammetric immunosensor based on molybdenum sulphide (MoS 2) and gold nanoparticles (Au NPs) for the determination of 25-hydroxy vitamin D 3 (25(OH)D 3) is reported. Anti-vit D (Ab-25(OH)D 3) was immobilized onto the cysteamine-modified MoS 2 and Au NPs which were deposited onto a fluoride tin oxide (FTO) electrode (Ab/Cys/Au/MoS 2/FTO). The MoS 2 sheets were prepared by hydrothermal method followed by an in situ growth of Au film onto the MoS 2/FTO surface. Self-assembled monolayer (SAM) of cysteamine was synthesized onto the Au/MoS 2/FTO which acts as a linker to covalently bind Ab-25(OH)D 3. The Ab-25(OH)D 3-immobilized Cys/Au/MoS 2/FTO was used to detect 25(OH)D 3 using differential pulse voltammetry. The electrochemical system provided an anodic peak current at a potential of +0.21 V vs. Ag/AgCl (satd. KCl) of ferricyanide/ferrocyanide redox couple. The detection principle relies on the inhibition of electron transfer at the electrode surface owing to the hindrance caused by the formation of immune complex between Ab-25(OH)D 3 and 25(OH)D 3. The immunosensor shows linear response from 1 pg mL −1 to 100 ng mL −1 25(OH)D 3 and a sensitivity of 189 μA [log (pg mL −1)] −1 cm −2 along with a low limit of detection (LOD) of 0.38 pg mL −1. The immunosensor is highly selective towards 25(OH)D 3 and presented a long shelf life of 28 days. Also, the immunosensor exhibits satisfactory performance towards spiked human serum samples with recovery between 95.1 and 102% (RSD 1.15–3.22%).