Objective To analyze the difference of sputum flora between acute exacerbation and stable chronic obstructive pulmonary disease (COPD) patients basing on metagenomic next generation sequencing (mNGS), and its relationship with clinical indicators. The role of sputum flora of COPD patients in unexplained deterioration was explored, so as to find a targeted treatment plan. Methods From December 2021 to June 2022, 54 COPD patients who had a history of smoking were recruited, including 25 patients in stable COPD (SCOPD group) and 29 patients in acute exacerbation (AECOPD group). The sputum was collected and sequenced by mNGS, and the difference of sputum flora between the two groups was compared. Results Compared with SCOPD group, the evenness of sputum flora (Shannon index) in AECOPD group decreased significantly (P=0.019, Mann-Whitney U test). At the phylum level, the relative abundance of Fusobacteria in AECOPD group was significantly lower than that in SCOPD group (Z=–2.669, P=0.008). At genus level, compared with SCOPD group, the relative abundance of Fusobacterium and Haemophilus in AECOPD group decreased significantly (Z=–3.062, P=0.002; Z=–2.143, P=0.032), and the relative abundance of Granulicatella increased significantly (Z=–2.186, P=0.029). At species level, the relative abundance of sputum Haemophilus parainfluenzae, Moraxella catarrhalis and Haemophilus influenzae in AECOPD group was significantly lower than that in SCOPD group (Z=–2.230, P=0.026; Z=–2.125, P=0.034; Z=–2.099, P=0.036). At the time of acute exacerbation of COPD, the relative abundance of Gemella in sputum was positively correlated with forced expiratory volume in first second/forced vital capacity (FEV1/FVC) and body mass index (r=0.476, P=0.009; r=0.427, P=0.021), which was negatively correlated with nutrition risk screening 2002 (r=–0.570, P=0.001). The relative abundance of Neisseria and Neisseria subflava was negatively correlated with GOLD grade (r=–0.428, P=0.020; r=–0.455, P=0.013). The relative abundance of Rothia aeria was posotively correlated with C-reactive peotein (r=0.388, P=0.038). Conclusions There are significant differences of sputum flora in phylum, genus and species level between stable and acute exacerbation COPD patients. The evenness of sputum flora in COPD patients in acute exacerbation is significantly lower than that in patients in stable stage. Fusobacteria, Fusobacterium, Gemella and Nesseria (Neisseria subflava) may play a beneficial role in COPD, while Rothia aeria may be associated with COPD exacerbation.