Abstract: Acetobacteraceae has garnered significant attention because of its unique properties and the broad applications of the bacterial cellulose it produces. However, unlike model strains, Acetobacteraceae have few synthetic biology applications because they are difficult to manipulate genetically and have insufficient genetic regulatory elements, among other factors. To address this limitation, this study characterized the fundamental properties and synthetic biology elements of three commonly used bacterial cellulose-producing strains. First, the basic characteristics of the three strains, including their cellulose film production ability, division time, antibiotic susceptibility, and plasmid features, were analyzed. Two inducible promoters (pTrc and pLux101) were subsequently characterized within the three strains. The inducibility of the pTrc promoter was relatively low across the three strains (induction ratio: 1.98–6.39), whereas the pLux101 promoter demonstrated a significantly greater level of inducibility within the three strains (induction ratio: 87.28–216.71). Finally, through gene knockout experiments, this study identified four genes essential for bacterial cellulose film production in the genome of the Gluconacetobacter hansenii ATCC 5358 strain. This study not only enriches the library of synthetic biology elements in nonmodel strains, but also lays the foundation for the synthetic biology applications of Acetobacteraceae.