In order to provide ports with more accurate and rapid detection methods for identifying three species of arawana in Osteoglossidae, including Scleropages formosus, Osteoglossum bicirrhosum, and Arapaima gigas, the COI and 16S RNA gene sequences were compared and analyzed in the three fish species and their related species. Three sets of specific primers and probes were designed and screened to establish a multiplex fluorescence PCR detection method that can simultaneously detect and identify the three arawana fish by optimizing the concentration of primers and probes and reaction system conditions. The sensitivity, repeatability, stability, and specificity of the method were evaluated separately. The results showed that the minimum detection limit was found to be 3.789×10^-6 ng/μL for S. formosus, 5.393×10^-6 ng/μL for O. bicirrhosum, and 4.229×10^-6 ng/μL for A. gigas, with 1×10^-6 ng/μL in this method. The relative coefficients r² of the standard curves for detecting three fish methods are all greater than 0.99, indicating a good linear relationship. The intra-batch coefficient of variation (CV) was ranged from 0.53% to 0.59%, and the inter-batch of CV from 0.26% to 0.48%, both below 2%, and without cross-reaction in specific detection for 13 species of ornamental fish with similar morphology and common characteristics imported through ports. In addition, 58 ornamental fish samples collected from ornamental fish farms and trading markets, as well as 37 imported ornamental fish from countries including Indonesia and Thailand, were identified with a 100% compliance rate, indicating that the method established in this study has high sensitivity, strong specificity, and stability, and can be used for the detection and identification of ornamental fish entering and exiting ports.

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