Recent developments in high-throughput sequencing (HTS) technologies have made it feasible to sequence the complete transcriptomes of non-model organisms or metatranscriptomes from environmental samples. The challenge after generating hundreds of millions of sequences is to annotate these transcripts and classify the transcripts based on their putative functions. Because many biological scientists lack the knowledge to install Linux-based software packages or maintain databases used for transcript annotation, we developed an automatic annotation tool with an easy-to-use interface. To elucidate the potential functions of gene transcripts, we integrated well-established annotation tools: Blast2GO, PRIAM and RPS BLAST in a web-based service, FastAnnotator, which can assign Gene Ontology (GO) terms, Enzyme Commission numbers (EC numbers) and functional domains to query sequences. Using six transcriptome sequence datasets as examples, we demonstrated the ability of FastAnnotator to assign functional annotations. FastAnnotator annotated 88.1% and 81.3% of the transcripts from the well-studied organisms Caenorhabditis elegans and Streptococcus parasanguinis, respectively. Furthermore, FastAnnotator annotated 62.9%, 20.4%, 53.1% and 42.0% of the sequences from the transcriptomes of sweet potato, clam, amoeba, and Trichomonas vaginalis, respectively, which lack reference genomes. We demonstrated that FastAnnotator can complete the annotation process in a reasonable amount of time and is suitable for the annotation of transcriptomes from model organisms or organisms for which annotated reference genomes are not avaiable. The sequencing process no longer represents the bottleneck in the study of genomics, and automatic annotation tools have become invaluable as the annotation procedure has become the limiting step. We present FastAnnotator, which was an automated annotation web tool designed to efficiently annotate sequences with their gene functions, enzyme functions or domains. FastAnnotator is useful in transcriptome studies and especially for those focusing on non-model organisms or metatranscriptomes. FastAnnotator does not require local installation and is freely available at http://fastannotator.cgu.edu.tw.