The total number of marine microorganisms is estimated to be 10³⁰. They are the key components driving marine energy and material recycling and maintaining ecological balance. Research advances in marine microbial diversity contribute to our understanding of the operative mechanisms of marine ecosystems. These knowledges can guide our respondence to ecological crisis of global oceans and our exploration of marine microbial resources. As the number of culturable marine microorganisms is currently low, our knowledges to the abundance, physiological characteristics and ecological functions of marine microbial communities are greatly limited. Ribosomal RNA sequencing technologies can rapidly and accurately identify or classify microorganisms based on nucleic acid sequences at low-cost. These technologies have been used in studying community structure and evolutionary and ecological relationships between microorganisms. In recent years, with the benefit of rapid development in sequencing technologies, marine microbial studies based ribosomal RNA sequencing analyses have achieved remarkable progresses in discovering novel marine microbial lineages, revealing ecological mechanisms and evolutionary relationships between marine microorganisms, discovering novel metabolites and applying in marine bioremediation. In this review, we introduce the technical principles of ribosomal RNA sequencing, the application of three generations of sequencing technologies in studying marine microbial diversity, and variable combinations of library construction and sequencing technologies. Finally, we address prospectives in utilizing these sequencing strategies in studies with different purposes.