Medium-chain alpha,omega-dicarboxylic acids produced from renewable long-chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer-Villiger monooxygenases are used in the production of omega-hydroxycarboxylic acids from long-chain fatty acids, the engineering of biocatalysts involved in the production of alpha,omega-dicarboxylic acids from omega-hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes,Micrococcus luteusalcohol dehydrogenase andArchangium violaceumaldehyde dehydrogenase, for the efficient production of alpha,omega-dicarboxylic acids from omega-hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40-fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium-chain alpha,omega-dicarboxylic acids from renewable long-chain fatty acids.