Saturated α-substituted β ketoesters are important building blocks in synthesis of pharmaceuticals and agrochemicals. Herein, we report a one-pot biosynthesis of α-substituted β ketoesters via Knoevenagel condensation and reduction of received unsaturated alkenes in situ, catalyzed by single ene-reductase (NerA). A series of inexpensive and readily available aldehydes and 1,3-diketones were condensed and reduced by NerA in aqueous solution at room temperature. We also note that low loadings (3 mg/ml) of NerA are sufficient to facilitate the cascade process, both E and Z isomeric intermediates can be reduced effectively and improved the overall yield up to 95%. Meanwhile, the method can be applied in preparative-scale synthesis of pharmaceutical intermediate. This process conforms to the concepts of green chemistry and shows advantages for synthesis of high value saturated α-substituted β ketoesters.