This study aims to establish a rapid and high-throughput bacterial species identification platform by utilizing colony PCR to efficiently amplify full-length 16S rDNA from bacterial strains, combined with a multiplex strategy and PacBio HiFi long-read sequencing technology to simultaneously complete sequencing and classification of multiple strains. This method simplifies the traditional workflow that requires individual DNA isolation and purification, thereby saving substantial labor and time while enhancing identification efficiency and throughput. Results show that under this platform, more than 75% of strains can be successfully cultured, and up to 83.2% of strains can be amplified and sequenced for 16S rDNA, demonstrating high stability and broad applicability. Compared with conventional identification approaches, this platform offers clear advantages such as shorter processing time, lower cost, simpler operation, and higher success rates, making it particularly suitable for microbial research scenarios requiring high-throughput identification. In addition to rapid identification of environmental microorganisms, this platform can also be widely applied in food safety testing, preliminary identification of clinical pathogens, agricultural soil microbiome analysis, classification and screening of biocontrol strains, as well as monitoring and management of industrial fermentation strains. In the future, with the further reduction of sequencing costs and the introduction of automated systems, this platform will have even greater potential for scalability and real-time applications, and is expected to become an indispensable core technology in both microbiological research and industrial applications.