To examine the pathogen diagnostic performance of targeted high-throughput next-gen sequencing (tNGS) in respiratory infectious diseases in preterm infants using dynamic follow-up.Clinical samples of respiratory secretions were consecutively collected from 20 preterm infants weekly for 5 weeks, during which 10 developed bronchopulmonary dysplasia. Pathogen identification from these collected specimens was performed by both conventional cultivation and tNGS.We found that targeted next-generation sequencing shared a 90.9% full or partial consistency for lower respiratory pathogen detection with the traditional culture-based approach, and increased the detection rate by 105.9%. Moreover, most of the pathogens identified by tNGS were diminished in patients after treatment.This study reveals the high sensitivity and performance of targeted high-throughput sequencing for respiratory infectious disease diagnosis and pathogen identification. The trial registry number is NCT03850457, and the trial URL is https://clinicaltrials.gov/ct2/show/NCT03850457.