The colonial ascidian Didemnum vexillum, sea carpet squirt, is not only a key marine organism to study morphological ancestral patterns of chordates evolution but it is also of great ecological importance due to its status as a major invasive species. Non-coding RNAs, in particular microRNAs (miRNAs), are important regulatory genes that impact development and environmental adaptation. Beyond miRNAs, not much in known about tunicate ncRNAs. We provide here a comprehensive homology-based annotation of non-coding RNAs in the recently sequenced genome of D. vexillum. To this end we employed a combination of several computational approaches, including blast searches with a wide range of parameters, and secondary structured centered survey with infernal. The resulting candidate set was curated extensively to produce a high-quality ncRNA annotation of the first draft of the D. vexillum genome. It comprises 57 miRNA families, 4 families of ribosomal RNAs, 22 isoacceptor classes of tRNAs (of which more than 72 % of loci are pseudogenes), 13 snRNAs, 12 snoRNAs, and 1 other RNA family. Additionally, 21 families of mitochondrial tRNAs and 2 of mitochondrial ribosomal RNAs and 1 long non-coding RNA. The comprehensive annotation of the D. vexillum non-coding RNAs provides a starting point towards a better understanding of the restructuring of the small RNA system in ascidians. Furthermore it provides a valuable research for efforts to establish detailed non-coding RNA annotations for other recently published and recently sequences in tunicate genomes.