Mucosal microbiomes play an important role in digestion, nutrition, and resistance to pathogens and toxins in fish; thus, characterizing the mucosal microbiomes of economically important fish species is paramount. In this study, mucosal microbiomes of healthy leopard coral grouper (Plectropomus leopardus), which is of economic importance worldwide, were systematically analyzed and compared by using 16S rRNA amplicon sequencing. The analysis of alpha-diversity and beta-diversity revealed significant differences in the structure and composition of the microbial community between different mucosal layers of P. leopardus. The skin microbiota showed the lowest microbial diversity among the three tissues, and the highest interindividual variability within gill groups was observed. Proteobacteria is the dominant phylum of the gut, skin, and gill microbiomes. Microbial biomarkers of the three mucosal tissues were identified, with the genera Parvibaculum, Aminobacter, Sphingobium, and Ralstonia for skin; Mesoflavibacter, Winogradskyella, Malaciobacter, Nautella, and Marinobacterium for gills; and Cetobacterium, Photobacterium, and Vibrio for gut. These genera were also the core microorganisms in each tissue. A large number of identical pathways of the three mucosal surfaces microbiome (e.g., pathways related to metabolism, human diseases, genetic/environmental information processing) and some specifically enriched pathways in each tissue were identified. Co-occurrence network analysis revealed that there were more ecological communities and higher functional diversity in the gill microbiota, and the interactions between gut microbiota are closer and more stable. Our study provides a new perspective for a comprehensive understanding of the mucosal microbiota of P. leopardus, contributing to an in-depth exploration of the interaction between fish and microorganisms, and may help to predict potential disease outbreaks, thus promoting the development of the fish farming industry.