Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor‐ and resource‐intensive for high‐throughput requirements. Currently, several instruments have been integrated for automated and high‐throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single‐cell microliter‐droplet screening system (MISS Cell), for automated, high‐throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high‐throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate‐producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high‐throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.