Background: The emergence of SARS-CoV-2 in late 2019 sparked the global COVID19 pandemic, leading to varied vaccine policies worldwide. The evolving patterns of respiratory pathogens, aside from SARS-CoV-2, during the pandemic have had a significant impact on the development of vaccine strategies. Methods: This study explores the landscape of respiratory pathogens, encompassing SARS-CoV2, respiratory syncytial virus (RSV), and influenza viruses, through a retrospective analysis of data obtained from the BioFire Respiratory Panel 2.1 (RP 2.1) at China Medical University HosResults: Among the 7950 respiratory samples studied, pediatric cases exhibited higher positivity (64. 9%, 2488/3835) and mixed detection rates (43 .8%, 1090/2488) than adults. Annual mixed detection rates increased (27.9-48%). Prevalence analysis revealed diverse patterns across age groups, with higher rates in pediatrics. Notably, human rhinovirus/enterovirus predominated (48.1%). Mixed detection illustrated viral co-detections, notably with parainfluenza viruses and adenovirus. Government policies and pandemic dynamics influenced infection patterns, with RSV resurgence after May 2022. Age-specific RSV detection demonstrated a shift, influencing vaccine considerations. Amid global vaccine initiatives, RSV''s increasing trend in adults warrants attention. Conclusions: This comprehensive analysis emphasizes the importance of multiplex PCR testing in shaping targeted vaccination strategies during evolving respiratory pathogen landscapes. Copyright (c) 2024, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).