Objective Combined the traditional isolation culture, ITS sequencing and metagenomics sequencing technology to establish a new method for the analysis of fungal community composition and species diversity in Pu'er tea at retail markets. Methods Pu'er tea samples were collected at retail markets and tested for cultivable fungi in accordance with “National food safety standard Food microbiological examination:Counting of Molds and Yeasts”(GB 4789.15—2016), and the isolated strains were sequenced by ITS and analyzed in the NCBI database; the fungal genomic DNA from Pu'er tea samples was extracted by CTAB method, and performed PCR amplification and amplicon sequencing; high-throughput sequencing data was used to build OTUs clustering, and carried out species composition and diversity analysis. Results Twelve fungi were isolated and cultured from Pu'er tea samples, mainly Aspergillus and Lichtheimia, of which Aspergillus niger had the highest proportion (25%, 3/12). The result of species distribution based on metagenomics showed that the diversity and uniformity of fungal species in Pu'er tea samples were low, and there were relatively high number of dominant species; the highest proportion of fungi was Ascomycota (74.03%), and at the genus level, the fungi in Pu'er tea were dominated by Blastobotrys (49.43%) while Aspergillus accounted for a relatively low percentage (14.04%). Conclusion This study comprehensively applied traditional isolation culture, molecular biology and high-throughput metagenomics sequencing technology to establish a new method for the analysis of fungal community composition and species diversity in Pu'er tea at retail markets. investigate the fungal community structure in Pu'er tea and whether it would contaminate toxin-producing strains such as Aspergillus flavus. It provided ideas and references for further research on the microbial community structure in Pu'er tea and its impacts on quality and safety.