In situ detection of functional genes is informative for understanding microbial physiology. Most methods of detecting functional genes employ multiple oligonucleotides or polynucleotide probes. However, single oligonucleotide probes are superior in terms of specificity and flexibility in probe design. Here we describe the detection of a single copy functional gene, the methyl coenzyme M reductase gene, in a methanogen by two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a single oligonucleotide probe without pre-amplification of target nucleic acids. Locked-nucleic-acid-incorporated DNA probes were employed to achieve high specificity and affinity. Although problems associated with non-removable nonspecific binding of the antibody could not be overcome completely, single copy gene detection was carried out with single mismatch descriminatable specificity; however, only around 15% of cells were detected. The detection rate increased when a multiple copy gene like rrn in Escherichia coli was targeted, indicating that a certain number of target molecules are necessary to achieve a high detection rate. Although possible applications of this technique to environmental samples remain restricted, the results presented the potential of gene detection by FISH with single oligonucleotide probes.