Both floral development and evolutionary trends of orchid flowers have long attracted the interest of biologists. However, expressed sequences derived from the flowers of other orchid subfamilies are still scarce except for a few species in Epidendroideae. In order to broadly increase our scope of Orchidaceae genetic information, we updated the OrchidBase to version 2.0 which has 1,562,071 newly added floral non-redundant transcribed sequences (unigenes) collected comprehensively from 10 orchid species across five subfamilies of Orchidaceae. A total of 662,671,362 reads were obtained by using next-generation sequencing (NGS) Solexa Illumina sequencers. After assembly, on average 156,207 unigenes were generated for each species. The average length of a unigene is 347 bp. We made a detailed annotation including general information, relative expression level, gene ontology (GO), KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping and gene network prediction. The online resources for putative annotation can be searched either by text or by using BLAST, and the results can be explored on the website and downloaded. We have re-designed the user interface in the new version. Users can enter the Phalaenopsis transcriptome or Orchidaceae floral transcriptome to browse or search the unigenes. OrchidBase 2.0 is freely available at http://orchidbase.itps.ncku.edu.tw/.