Recombinant adeno-associated viruses (rAAVs) play a pivotal role in gene therapy, yet the molecular interactions underlying their production in host cells remain incompletely understood. Non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs), are increasingly recognized as key regulators of viral and cellular processes. This study investigates the dynamic expression profiles of miRNAs and snoRNAs during rAAV plasmid transfection and vector production in HEK293F cells. A total of 142 miRNAs were differentially expressed during the peak phase of rAAV production, with 128 associated with the Gene Ontology term ‘viral process’, indicating broad involvement in host-virus interactions. Target gene analysis linked these miRNAs to biological pathways such as nucleocytoplasmic transport, innate immunity, apoptosis, and transcriptional regulation, highlighting their potential roles in shaping the cellular environment during viral vector assembly. In contrast, snoRNAs exhibited more modest changes in expression, yet five were significantly differentially expressed during active production, suggesting a possible, underexplored involvement in viral replication. These findings illuminate the underexplored contributions of ncRNAs to the host response during rAAV biogenesis and provide a valuable resource for understanding how cellular regulatory networks are engaged throughout vector production.