This study focused on a new plasmid and new recombinant strain developed for the production of microbial chondroitin sulfate a new and limited area of study the strategies we applied for the production of microbial chondroitin sulfate, and the possible contributions of this study to published research literature. In this study, pETM6-PACF, which carries the genes responsible for capsular chondroitin synthesis [kfA, kfoC, kfoF] was used as the basic plasmid. The Vitreoscilla hemoglobin gene region was transformed into this basic plasmid and the common expression of both gene groups was added to research literature for the first time. This plasmid was transferred to non-pathogenic E. coil (C2987) to produce a completely new chondroitin source specific to this study. Following the transformation by chondroitin synthesis, and the subsequent microbial production of chondroitin by the application of purification protocols, microbial chondroitin sulfate was produced in sulfate form. Consequently, in comparison to published literature, a product with a low molecular weight value of 269 Daltons was developed. This product, which has significant potential drug potency, can be used in many different areas as a novel and unique biomedical product.