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Dual detection of Angelica sinensis and Angelica acutiloba was performed by a combined method of ladder-shape melting temperature nucleic acid isothermal amplification technique (LMTIA) and proofreading enzyme-mediated probe (Proofman). The optimum temperature, sensitivity, and stability of the primers for Angelica sinensis and Angelica acutiloba were determined using distilled water as the negative template and Angelica sinensis and Angelica acutiloba genomic DNA as the positive template, and real samples were tested. It was proved that the Proofman-LMTIA dual detection of Angelica sinensis and Angelica acutiloba had an optimal temperature of 62 °C, and the sensitivity of the primers was as low as 10 pg/µL for Angelica sinensis and 1 pg/µL for Angelica acutiloba, with good stability. Seven commercial Angelica sinensis samples were tested and all were identified as Angelica sinensis. This study provides a new detection method for the identification of Angelica sinensis and Angelica acutiloba. [ABSTRACT FROM AUTHOR]