Abstract: To screen for key RNA interference (RNAi) target genes for controlling Plutella xylostella, trehalase (PxTRE), vacuolar ATP synthase subunit E (PxATPE), ribosomal protein S13 (PxRPS13), and chitinase (PxCHT) were selected. Multiple RNA microspheres (RMS) were designed and synthesized using rolling circle transcription (RCT) technology. After administering the RMS to third-instar larvae via oral feeding, larval development and mortality were assessed. The results showed that gene silencing was significant after 48 hours of treatment. Compared to the RMS-EGFP control group, mRNA expression levels of PxTRE, PxATPE, PxRPS13, and PxCHT were reduced by 74.84%, 73.36%, 62.90%, and 38.10%, respectively (P < 0.05). In addition, multi-RMS RMS-PxTA（RMS-PxTRE/ATPE） and RMS-PxTC（RMS-PxTRE/CHT）significantly downregulated the expression of the corresponding genes. In the RMS-PxTA treatment, the expression levels of PxTRE and PxATPE were reduced by 86.16% and 96.38%, respectively (P < 0.05). Similarly, in the RMS-PxTC treatment, the expression levels of PxCHT were decreased by 45.22% and 28.02%, respectively (P < 0.05) . After 72 hours, the larval mortality rates in the RMS-PxTRE、RMS-PxATPE、RMS-PxCHT和RMS-PxRPS13、RMS-PxTA and RMS-PxTC treatments were 62.22%, 86.67%, 60.00%, 57.78%, 35.56%, and 66.67%, respectively. Surviving larvae in the treatment groups exhibited reduced feeding, shortened body length, and molting failure. These findings suggest that RCT technology is a viable method for rapid screening of RNAi target genes in P. xylostella. Among the tested target genes, PxATPE demonstrates potential as a highly effective target for RNA-based biopesticide, offering promising prospects for environmentally friendly control of P. xylostella.