Vitamin D plays a vital role in health; therefore, there is a need for a sensitive, selective, quick, and easy technique for its determination. Previous research has proposed electrochemical biosensors based on different carbon materials that are functionalized with various electrochemical biosensors. However, the existing problems and future opportunities for these sensors need further research. The practical use of electrochemical biosensors for vitamin D detection is attributed to their ability to detect vitamin D from diverse samples, including vitamin D production, in nature. This chapter provides recent investigations on the utilization of electrochemical biosensors for vitamin D detection such as Ab-25OHD/SPE/FMTAD, CYP27B1/GCE, SiO2/GO/Ni(OH)2/GCE, BSA/Ab-VD2/CD-CH/ITO, BSA/Anti VD/Fe3O4 PANnFs/ITO, BSA/Ab-VD/Asp-Gd2O3NRs/ITO, 25OHD, 25OHD Antibody, IoT-Enabled Enzyme Embossed Biosensor, Au-Pt NPs/APTES/FTO, and GCN-β-CD/Au nanocomposite. The chapter aims to provide a comprehensive overview of the recent developments in electrochemical biosensors for accurate and efficient vitamin D detection.