Abstract: Abstract Objective: To explore the clinical application value of Nanopore metagenomics sequencing in the rapid detection of pediatric respiratory tract pathogens. Methods: We collected a bronchoalveolar lavage fluid sample from a severe pneumonia patient, which was positive for adenovirus antigen by conventional tests and Mycoplasma pneumoniae and Epstein-Barr virus by PCR. The patient was discharged after recovery. After DNA extraction, Nanopore and Illumina sequencing were performed respectively. We constructed microbe reference genome database and developed real-time analysis pipeline for Nanopore sequencing data. Nanopore and Illumina sequencing data were mapped to the reference database to detect the pathogens. Results: From receipt of sample to the detection of adenovirus, Nanopore, Illumina and conventional methods took 3.2 hours, 55.5 hours and 48 hours respectively, and cost 4,200, 3,600 and 1,790 RMB respectively. We detected 1,093 adenovirus reads at 12 minutes after the start of Nanopore sequencing. A total of 4,920,000 reads were sequenced. Among them, we detected 99,284 adenovirus reads, accounting for 2.02% of all the sequencing reads. In addition to adenovirus, Mycoplasma pneumoniae and Epstein-Barr virus reads were also detected. The same sample was validated by Illumina sequencing and the detected microbial composition was similar to Nanopore. Conclusion: Nanopore metagenomics sequencing could rapidly and real-timely detect all potential pediatric respiratory track pathogens.