In this report, CuBi2O4/Co3O4 has been used for the electrochemical detection of glucose. Herein, CuBi2O4 nanorods and Co3O4 has been hybridized with CuBi2O4 to form CuBi2O4/Co3O4 nanocomposite hydrothermally. CuBi2O4 and CuBi2O4/Co3O4 electrodes are fabricated by using drop casting method. Structural, morphological studies of CuBi2O4/Co3O4 and electrochemical characterizations pertaining to its performance in comparison to CuBi2O4 for electrochemical detection of glucose are performed and discussed elaborately. The detailed mechanism involved in the electrochemical detection of glucose is proposed. The electrode has shown the sensitivity of 49 μA mM−1 cm−2 with a limit of detection (LOD) of 0.136 μM. DFT calculations are performed on the optimized molecules involved in the electrochemical detection of glucose confirming CuBi2O4/Co3O4 acting as electron donor while glucose as electron acceptor. Further, natural bond orbital (NBO) for charge distribution calculations and ESP mappings for the optimized molecules are performed to understand nucleophilic/electrophilic positions. This study reveals that CuBi2O4 and its modifications are promising materials for the electrochemical detection of glucose. Further, the role of Co3O4 in improving the sensitivity and electrochemical performance for detection of glucose has been discussed. © 2022 Wiley-VCH GmbH.