6-Hydroxyflavone (6HF) contains attractive biological properties of significance pharmacologically and has been discovered as an effective diabetic medication. However, due to its high degradation in aqueous solution, its efficacy in biological treatment remains a considerable obstacle. Thus, conjugating a polymer, polyethylene-glycol, to 6HF by direct esterification between the carboxyl group of PEG and the hydroxyl group at the sixth carbon of the 6HF biomolecule is one of the approaches applied in this research to increase its stability while maintaining the inherent biological characteristics. This study examined the optimum esterification reaction conditions for conjugate PEG-6HF utilizing EDC and DMAP as conjugation reagents in various solvents, such as DMSO, PBS, and PBS 10 mM HEPES pH7.4 with the assistance of HOBt, including its stability in the biomimicking environment. For this purpose, PEG-6HF connected through the ester bond was validated using various analytical techniques such as FTIR, UV-Vis spectroscopy, and HPLC. , Notably, esterification at 25 °C for 24 hours in a 10 mM HEPES pH 7.4 buffer solution using EDC with HOBt resulted in the most significant conjugation percentage, 42 percent. Furthermore, PEG-6HF revealed 1.3 times lower degradation percentages of 6HF biomolecules than unconjugated-6HF following 6 hours of incubation in 10 mM HEPES pH 7.4 at 37 °C. Hence, the optimal conditions and the resulting conjugation percentage with high stability are expected to be a fundamental approach to conjugated polymer with a biomolecule.