Background: Respiratory infectious viruses, such as the novel coronavirus SARS-CoV-2, are highly transmissible and undergo rapid genetic evolution, which leads to the emergence of multiple subtypes with diverse mutation patterns. However, detecting and differentiating between these subtypes present significant challenges in the field of respiratory virus surveillance. Methods: To address these challenges, we developed a novel detection approach that integrates amplification refractory mutation system PCR (ARMS-PCR) with molecular beacon probes. The ARMS-PCR primers were designed to selectively amplify specific subtypes by targeting adjacent mutation sites, while the molecular beacon probes allowed for further discrimination of the amplified products. This combined approach effectively addressed the issues of non-specific binding and improved detection accuracy. Results: Our method demonstrated high specificity and sensitivity in the identification and differentiation of respiratory virus subtypes. Using real-time fluorescence PCR, we achieved a detection limit of approximately 106 copies/mL. Moreover, through the direct analysis of fluorescence signals, we further enhanced the sensitivity to a detection limit of 104 copies/mL. This robust and accurate detection approach is capable of identifying and differentiating between respiratory virus subtypes, including those with complex mutation patterns. Conclusions: The integration of ARMS-PCR and molecular beacon probes is a reliable and efficient solution for the rapid and precise monitoring of evolving respiratory infectious diseases, and it has the potential to facilitate early diagnosis and effective control measures. Further research is needed to expand the application of this detection method to other respiratory viruses and optimize its workflow for clinical and public health settings.