MicroRNAs (miRNAs) are short noncoding RNAs (20–25 nucleotides) that regulate gene expression posttranscriptionally. However, identification and characterization of miRNAs remain limited for conifer species. In this study, we applied transcriptome-wide miRNAs sequencing to a conifer species Platycladus orientalis, which is highly adaptable to a wide range of environmental adversities, including drought, barren soil, and mild salinity. A total of 17,181,542 raw reads were obtained from the Illumina sequencing platform; 31 conserved and 91 novel miRNAs were identified, and their unique characteristics were further analyzed. Ten randomly selected miRNAs were validated by quantificational real-time polymerase chain reaction. Through miRNA target predictions based on psRNATarget, 2331 unique mRNAs were predicted to be targets of P. orientalis miRNAs that involved in 187 metabolic pathways in KEGG database. These targets included not only important transcription factors (e.g., class III homeodomain leucine zipper targeted by por-miR166d) but also indispensable nontranscriptional factor proteins (i.e., por-miR482a-3p regulated nucleotide-binding site leucine-rich repeat protein). Interestingly, six miRNAs (por-miR16, -miR44, -miR60-5p, -miR69–3p, -miR166b-5p, and -miR395c) were found in adaptation-related pathways (e.g., drought), indicating their possible involved in this species’ stress-tolerance characteristics. The present study provided essential information for understanding the regulatory role of miRNAs in P. orientalis and sheds light on their possible use in tree improvement for stress tolerance.