Desulfovibrio spp. is predominant member of sulphate‐reducing bacteria in human gut microbiota. Previous studies indicated that the isolation of Desulfovibrio strains from human faecal samples is very important to study the roles of human intestinal Desulfovibrio spp. in maintaining healthy states or causing diseases, as well as defining their biological characteristics. However, there are very few reports describing the isolation of Desulfovibrio spp. from human faecal samples. In this study, faecal samples were inoculated into various media containing different components. The enriched culture communities were identified using 16S rRNA gene high‐throughput sequencing analysis, enabling us to identify the specific components that enable the enrichment of Desulfovibrio. Using this information, we developed five specific media and identified an effective enrichment medium that produced the highest relative abundance of Desulfovibrio in communities cultured from four faecal samples (26·5, 73·5, 44·7 and 77·6% respectively). In addition, the major non‐Desulfovibrio genera were identified. Finally, three species of Desulfovibrio, D. desulfuricans, D. piger and D. legallii were isolated, representing the first time that has D. legallii been isolated from a human gastrointestinal source. Significance and Impact of the Study ost of the human intestinal bacteria have not been cultured because of lack of appropriate culture method and appropriate media. Desulfovibrio spp. is associated with several clinical conditions like inflammatory bowel disease, but until now there are very few reports describing the isolation of Desulfovibrio spp. from human faecal samples. In this study, 16S rRNA gene high‐throughput sequencing analysis was applied to screen appropriate enrichment media and selective cultivation of Desulfovibrio. This sequencing‐based directed culture method described here can be used for the selective cultivation of gut bacteria of interest. Significance and Impact of the Study: Most of the human intestinal bacteria have not been cultured because of lack of appropriate culture method and appropriate media. Desulfovibrio spp. is associated with several clinical conditions like inflammatory bowel disease, but until now there are very few reports describing the isolation of Desulfovibrio spp. from human faecal samples. In this study, 16S rRNA gene high‐throughput sequencing analysis was applied to screen appropriate enrichment media and selective cultivation of Desulfovibrio. This sequencing‐based directed culture method described here can be used for the selective cultivation of gut bacteria of interest.