Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides (nt). LncRNAs have high spatiotemporal specificity, and secondary structures have been preserved throughout evolution. They have been implicated in a range of biological processes and diseases and are emerging as key regulators of gene expression at the epigenetic, transcriptional, and post-transcriptional levels. Comparative analyses of lncRNA functions among multiple organisms have suggested that some of their mechanisms seem to be conserved. Transcriptome studies have found that some Drosophila lncRNAs have highly specific expression patterns in embryos, nerves, and gonads. In vivo studies of lncRNAs have revealed that dysregulated expression of lncRNAs in Drosophila may result in impaired embryo development, impaired neurological and gonadal functions, and poor stress resistance. In this review, we summarize the epigenetic, transcriptional, and post-transcriptional mechanisms of lncRNAs and mainly focus on recent insights into the transcriptome studies and biological functions of lncRNAs in Drosophila.