Abstract: Microorganisms are ubiquitously distributed across diverse ecological environments on Earth, especially in marine ecosystems, where they play a key role in the global material and energy cycle and hold immense potential. Exploring the community structure and physiological characteristics of microorganisms in various environmental media is fundamental to understanding their functional mechanisms in biological processes, and serves as a prerequisite for harnessing Earth’s microbiome to benefit humanity. However, conventional methods of laboratory culture and high-throughput nucleic acid sequencing fail to effectively distinguish dead cells, dormant cells and active microbial cells in environmental media, which impedes the accurate exploration of microbial functions to a certain extent. Recent advancements in next-generation physiological function detection technologies, particularly bioorthogonal non-canonical amino acid tagging (BONCAT), offer a robust solution to this challenge. BONCAT is characterized by its cost-effectiveness, simplicity, compatibility with standard laboratory instruments, and high adaptability to downstream analytical methods. Combined with diverse downstream techniques, BONCAT has broad application prospects in marine microbial research and is of great significance for revealing the changing composition and material conversion metabolism of active microbial communities in marine ecosystems. Specifically, BONCAT-flow cytometry (BONCAT-FCM) enables rapid quantitative analysis of microbial activity; BONCAT-fluorescence in situ hybridization (BONCAT-FISH) facilitates in situ identification of active target microorganisms; and BONCAT-fluorescence-activated cell sorting (BONCAT-FACS) allows the isolation of highly active microbial subpopulations for in-depth functional analyses. Nevertheless, the current application of BONCAT in marine microbial research faces limitations. To enhance the accuracy of microbial activity and functional analyses, future efforts should focus on elucidating the mechanisms of BONCAT substrate entering cells to optimize reaction conditions, refining BONCAT-FACS workflows and sample pretreatment protocols to enable proteomic and transcriptomic analyses of low-biomass samples, and advancing the development of novel BONCAT substrates to improve reaction efficiency and anti-interference of BONCAT reactions. Therefore, we suggest that interdisciplinary collaboration among microbiologists, chemists, and engineers be established to propel innovation in this field, thereby unlocking new opportunities for exploring and harnessing microbiomes across diverse ecosystems.