[Aim] MicroRNAs (miRNAs) play important regulatory roles in insect growth and development. In this study, miRNAs preferentially expressed in Plutella xylostella larvae were identified, and their potential functions in growth and development were explored. [Method] The small RNA libraries of the eggs, 3rd instar larvae, pupae, and adults of P. xylostella were sequenced. Based on bioinformatic analysis, miRNAs preferentially expressed in the larvae were selected and verified by quantitative reverse transcription-PCR. The target genes of the miRNAs were predicted using RNAhybrid and miRanda. miRNAs with potential target genes related to growth and development were selected, and an in vitro dual luciferase reporter assay was conducted to test miRNA-mRNA interactions. [Result] A small RNA library constructed from the four developmental stages of P. xylostella was sequenced on an Illumina sequencing platform, revealing 2116 known and 189 novel miRNAs. A total of 265, 228, and 132 miRNAs showed significantly higher expression in larvae than in eggs, pupae, and adults, respectively. Further selection of 11 miRNAs that were highly expressed in the larvae was verified by quantitative reverse transcription-PCR, which exhibited a larval-based pattern and was consistent with the results of small RNA sequencing. Target gene prediction of these miRNAs showed that seven miRNAs may target the juvenile hormone metabolism pathway. In vitro analysis of the miRNA-mRNA interactions illustrated that pxy-miR-6094-3p, pxy-miR-750-3p, novel-m502, and miR-274 can target juvenile hormone esterase genes. [Conclusion] Larval-based miRNAs of P. xylostella may be involved in juvenile hormone metabolism regulation.