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Molybdenum (Mo) is an essential trace element for higher plants. Mo enzyme biosynthesis is regulated by several plant genes. A hydroponic trial was conducted to investigate the effect of Mo deficiency on the expression of genes that are related to Mo enzyme biosynthesis in winter wheat. The results showed that Mo deficiency decreased the activities of nitrate reductase (NR), sulphite oxidase (SO), aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) in the leaves or roots of winter wheat by regulating the expression of NR, SO, AO and XDH genes. Mo deficiency induced the expression of TaCnx2 and TaCnx5, which might result in the accumulation of cyclic pyranopterin monophosphate (cPMP) and molybdopterin (MPT) in wheat. Mo deficiency significantly decreased TaCnx1 expression, which is expected to reduce molybdenum cofactor (Moco) formation. Moco shortage induced the expression of TaAba3 in winter wheat. The blockade of Moco biosynthesis under conditions of Mo deficiency might decrease the activities of Mo enzymes.