Dodecanedioic acid (DDA) is highly useful to the chemical industry as a versatile precursor for producing the polyamide nylon-6,12, which is used for many technical applications, such as heat and chemical-resistant sheaths. However, DDA synthesis has several drawbacks, such as high energy input and cost-intensive removal of by-products. Therefore, alternative bio-based production routes are required due to increasing industrial demand for green chemicals and renewable products. Candida tropicalis converts petrochemical-based n-dodecanes to the corresponding dicarboxylic acids by targeted functionalization. To increase sustainability of the DDA production process, we tested dodecanoic acid methyl ester, which can be easily obtained from transesterification of coconut oil, in whole-cell biotransformation by C. tropicalis. By modifying selected process parameters, a final DDA concentration of 66 g/L was achieved using a highly reliable, small-scale bioreactor system. Crucial process development included a gradual pH shift, an optimized substrate feeding strategy, and monitoring the transcriptional profile.