Pinoresinol is a natural lignan with a high market value that has potential pharmacological and food supplement applications. Pinoresinol is currently isolated from plants， which suffers from low efficiency and yield. To produce pinoresinol from inexpensive and industrially available eugenol， an in vivo enzymatic cascade composed of vanillyl alcohol oxidase and peroxidase was designed， which scavenges H O automatically and eliminates protein purification and cofactor addition. Two peroxidases were screened and identified from Escherichia coli BL21 (DE3)， and tested in the enzymatic cascade. To balance the flux， different genetic architectures were constructed by using ePathBrick and fusion gene approaches. Scavenging H O alleviated by-product toxicity and enzyme inhibition， and led to efficient pinoresinol production. Optimization of the reaction conditions achieved a titer of 11.29?g/L pinoresinol. The molar yield and productivity were 52.77% and 1.03?g/(L?×?h)， respectively. The elegant strategy developed herein utilizes the harmful by-product to drive the biosynthetic reaction forward and simultaneously detoxify cells， thereby preventing enzyme inhibition. Biotechnol. Bioeng. 2017;114: 2066-2074. ? 2017 Wiley Periodicals， Inc.