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<i>Burkholderia cepacia</i> complex (BCC) contamination has resulted in recalls of non-sterile pharmaceutical products. The fast, sensitive, and specific detection of BCC is critical for ensuring the quality and safety of pharmaceutical products. In this study, a rapid flow cytometry-based detection method was developed using a fluorescence-labeled oligonucleotide <i>Kef</i> probe that specifically binds a KefB/KefC membrane protein sequence within BCC. Optimal conditions of a 1 nM <i>Kef</i> probe concentration at a 60 °C hybridization temperature for 30 min were determined and applied for the flow cytometry assay. The true-positive rate (sensitivity) and true-negative rate (specificity) of the <i>Kef</i> probe assay were 90% (18 positive out of 20 BCC species) and 88.9% (16 negative out of 18 non-BCC), respectively. The detection limit for <i>B. cenocepacia</i> AU1054 with the <i>Kef</i> probe flow cytometry assay in nuclease-free water was 1 CFU/mL. The average cell counts using the <i>Kef</i> probe assay from a concentration of 10 μg/mL chlorhexidine gluconate and 50 μg/mL benzalkonium chloride were similar to those of the RAPID-B total plate count (TPC). We demonstrate the potential of <i>Kef</i> probe flow cytometry as a more sensitive alternative to culture-based methods for detecting BCC in non-sterilized pharmaceutical raw materials and products with regards to water-based environments.