To study the role of polymerase chain reaction-high-resolution melt (PCR-HRM) in the detection of mixed bacteria from respiratory tract, the fluid from the lower respiratory tract of patients with chronic obstructive pulmonary disease (COPD) was collected through bronchoscopy. Total DNA of bacteria was extracted and the full-length 16S rDNA was amplified by PCR with 16S primers 27F/1492R. PCR products were electrophoresed and purified, and then connected to pGEMT-Easy carrier to build 16S rDNA clone library. The cloned library was amplified by 338F/518R (16S rDNA-V3 region universal primer). PCR-HRM in V3 region was performed by Roche LightCycler 480 real-time fluorescence quantitative PCR system. The data were analyzed by HRM GeneScan analysis software. According to the different HRM patterns, clonal strains were selected for sequencing to identify bacterial strain. The results showed that PCR-HRM could be responsive to distinguish the 16S rDNA-V3 regions from different bacteria of the lower respiratory tract and cloned strain sequencing sample size was significantly reduced according to HRM patterns. PCR-HRM technology can be used as a rapid, high-throughput, sensitive and economic detection method of bacterial diversity.