Chitosan is one of the most popular biopolymers used for biomedical applications with its unique properties of blood clotting and adhesion to tissues. Catechol-functionalized chitosan (CatChit) has shown a significant improvement of those properties of chitosan as biomaterials. However, some well-cited methods of CatChit preparation in existing literature, repeatedly followed by numerous research groups in the past decades, have not stressed the importance of the vulnerability of catechol to oxidation, which resulted in many priceless in vivo studies that used wrong materials, i.e., partially oxidized forms of CatChit. Since some key synthesis parameters were erroneous in those previous reports, it is a challenge to reproduce the published results. To avoid the loss of critical details with these repeated citations, it is essential that we re-establish the critical parameters in these methods. In this study, we examined the accuracy of existing protocols, and optimized one of the protocols to synthesize CatChit. We have confirmed that a notable degree of catechol oxidation is inevitable with the inaccurate synthetic protocols and the maintenance of pH < 5 throughout the preparation of CatChit is essential. We have also re-evaluated interaction between CatChit and mucin, which is widely present in the gastrointestinal (GI) tract, at different pH values using CatChit prepared via our optimized synthetic protocol. Turbidimetric titrations suggested that regardless of the reaction pH, the association between CatChit and mucin increased with increasing concentration of polymer with respect to mucin. The decrease in the average size of the aggregated particles observed by Dynamic Light Scattering (DLS) studies was attributed to the formation of a large number of aggregations with increasing polymer to mucin ratio. zeta potential (ZP) measurements suggested that at acidic reaction pH, the average particle size was dictated by electrostatic interactions, while at a physiological pH, consolidation of covalent and charge-based interactions contributed to the overall surface charge.