Display omitted •Chemical composition and functional activities of known essential oils.•Xanthine Oxidase bovine milk is more advantageous enzyme source in terms results, and savings.•Double enzyme detection is an innovative method approved its efficacy and quickness. In pharmaceutical labs, the major sources of xanthine oxidase enzyme (XO) used for the gout disease experiment are animals and humans. The aim of this study is to find the lucrative source for the experiment by the evaluation in vitro and in silico of the inhibitory activity of five essential oils (EOs), against human and bovine milk xanthine oxidase (HXO and BXO), using molecular docking and an innovative analysis method based on double enzyme detection (DED), investigated in this work for the first time. The DED method proved its efficacy, sensitivity, and quickness. The results show that the five EOs give an important inhibition to BXO and HXO with an IC50 of 3.67 ± 0.17 μg/ml, 3.89 ± 0.11 μg/ml, 3.76 ± 0.18 μg/ml, 2.37 ± 0.23 μg/ml, 3.43 ± 0.56 μg/ml for HXO, and to BXO with an IC50 of 6.26 ± 0.92 μg/ml, 5.53 ± 0.82 μg/ml, 10.59 ± 1.59 μg/ml, 1.74 ± 1 μg/ml, 5.33 ± 0.13 μg/ml, for respectively, cumin (Cuminum cyminum L.), fennel (Foeniculum vulgare Mill.), coriander (Coriandrum sativum L.), anise (Pimpinella anisum L.), and caraway (Carum carvi L.) EOs. In silico study based on molecular docking using autodock vina program was carried out to study the BXO and HXO inhibition mechanism and involved interactions for the first time. The results show that the five used EOs give an important and similar inhibition effect against XO with both enzyme sources, therefore we propose the bovine milk as a new economic enzyme source for lab experiment, which can promote a new, approach in future gout treatment.