Respiratory tract infection is one of the most prevalent medical conditions. Rapid and accurate diagnosis of pathogens is crucial prerequisite for precise and effective treatment. However, the sensitivity of traditional microbiological and immunological techniques for respiratory pathogens detection is low. The development and clinical applications of nucleic acid testing technology have dramatically improved the diagnostic ability of respiratory pathogens. The rational usage of appropriate nucleic acid testing techniques, based on the underlying diseases, the type of respiratory tract infections, and pathogen spectrum, as well as correct understanding its diagnostic significance, has become an important clinical problem. For this, the Committee of Chinese Laboratory Medical Education initiated a multi-disciplinary Chinese expert consensus. Basing on the both domestic and international guidelines and literature, the expert consensus conducted an analysis of the clinical application scenarios, technical characteristics, and performance validation requirements pertaining to real-time fluorescence polymerase chain reaction (PCR), isothermal amplification technology, digital PCR, nucleic acid point-of-care testing, and metagenomics Next Generation Sequencing. Furthermore, the consensus of experts also examined the wide-ranging applications of these techniques in diagnosing and managing respiratory tract infections, including acute upper respiratory tract infections, tracheobronchitis, community-acquired pneumonia, hospital-acquired pneumonia/ventilator-associated pneumonia, acute exacerbation of chronic obstructive pulmonary disease, tuberculosis, and respiratory infections in immunocompromised adults. We hope the consensus will provide helpful reference to the clinicians.