25‐Hydroxyvitamin D3 as a main circulating metabolite of vitamin D is usually measured in serum to evaluate the vitamin D status of humans. So, developing an accessible, fast response, sensitive, and selective detection method for 25‐hydroxyvitamin D3 is highly important. In this study, we designed a sensitive and selective electrochemical sensor based on the modification of glassy carbon electrode by nanocomposite of CuCo2O4/nitrogen‐doped carbon nanotubes and phosphorus‐doped graphene oxide. Then 25‐hydroxyvitamin D3‐imprinted polypyrrole was coated on the electrode surface through electropolymerization. Moreover, ferricyanide was used as a mediator for the creation of a readable signal, which was considerably decreased after rebinding of 25‐hydroxyvitamin D3 on the electrode. The proposed sensor successfully detected 25‐hydroxyvitamin D3 in the range of 0.002–10 μM, with a detection limit of 0.38 nM, which was highly lower than deficiency concentration (20 ng/ml; 49.92 nM). Finally, the proposed sensor was checked for detection of 25‐hydroxyvitamin D3 in serum samples with recovery in the range of 80%–106.42%. The results demonstrated the applicability of the designed sensor for the detection of 25‐hydroxyvitamin D3 in biological samples. Application of CuCo2O4/N‐doped CNTs/P‐doped GO nanocomposite and molecularly imprinted polymer for electrochemical detection of 25‐hydroxyvitamin D3.