α,ω-Dicarboxylic acids (α,ω-DCAs) have numerous applications, as raw materials for producing various commodities and polymers in chemical industry. For example, dodecanedioic acid (DDDA), a monomer of nylon 612 polymer is used for numerous applications such as plasticizers and adhesive. In this study, firstly the enzymatic production of DDDA from corresponding v-hydroxy fatty acid was optimized using aldehyde reductase (AHR) and aldehyde dehydrogenase (ALDH), and the cofactor imbalance and proficient regeneration thereof, is a major hurdle for the effective biosynthesis. To circumvent this, NAD (P)H oxidases (NOXs) from Lactobacillus brevis (LbrNOX), Brevibacterium glutamicum (BreNOX) and Lactobacillus reuteri (LreNOX) were examined for the AHR/ALDH/NOX cascade and LreNOX was identified as suitable enzyme. Moreover, LreNOX was fused with AHR and employing this fusion protein in Escherichia. coli (E. coli) holding ALDH, nearly complete conversion (>99%) into DDDA was achieved from 30 mM of 12-hydroxy dodecanoic acid within 12 h of whole-cell biotransformation (2.2-fold improvement in productivity) turning out to be beneficial. Finally, the synthesis of DDDA from corresponding free fatty acid (dodecanoic acid), was performed by employing combination of AHR/ ALDH/NOX cascade with CYP153AL.m, where 2.3 mM (0.53 g/L) of DDDA was produced in one-pot one step.