Biofilm formation is a well-known pathogenic mechanism in infections caused by Acinetobacter baumannii. Recently, a biofilm synthesis-associated gene has been found in A. baumannii ATCC19606. Bioinformatic analysis showed 2 transmembrane structures and an hmsS superfamily domain, which was related to biofilm formation. What is more, high homology sequences of the bfs gene were only present in A. baumannii spp., and the similarities of nucleotide sequences of the bfs gene from A. baumannii strains ATCC17978, ACICU, S1, AB307-0294, and AB0057 compared with the reported sequence of bfs (GenBank accession No.: NZ_GG704572) were all above 95%. The distribution and conservation of the bfs gene from clinically derived A. baumannii strains were verified through conventional polymerase chain reaction (PCR). After this, we established a bfs gene-based real-time quantitative PCR assay to detect A. baumannii. Species specificity and sensitivity assays were designed and validated. By using this method, all the A. baumannii strains separated from clinical samples were identified and showed good accordance with the results from biochemical identification. This study is the first report of developing a bfs gene-based quantitative polymerase chain reaction for rapid, stable, and specific detection of A. baumannii. This method can be applied to clinical laboratory diagnosis, and detection of A. baumannii present on medical instruments.