This review provides a comprehensive guide to researchers on the selection and application of microfluidic chip technologies for single-cell isolation and analysis. It highlights the design principles, separation mechanisms, chip characteristics, and cellular effects of various microfluidic chips, emphasizing their implications for subsequent multiomics and exosome analyses. The review also discusses the current challenges and future prospects of microfluidic chip technology in multiplex single-cell isolation and multiomic and exosome analyses. Single-cell multiomic and exosome analyses are crucial tools in various fields, including cancer research, immunology, neuroscience, microbiology, and drug development. Single-cell isolation is essential for reliable cell analysis, and microfluidic chips offer efficient and high-throughput solutions. The review covers the advantages and limitations of traditional single-cell isolation techniques, such as limiting dilution, laser capture microdissection (LCM), micromanipulation, and fluorescence-activated cell sorting (FACS), and introduces the benefits of microfluidic chips. The review discusses four main types of microfluidic chips for single-cell isolation: high-activity culture and multi-factor detection microchamber chips, high-efficiency and low-throughput double-layer valve chips, high-throughput and high-efficiency microdroplet chips, and high-throughput and high-efficiency microwell chips. Each type is evaluated based on its working principles, separation mechanisms, structural characteristics, and performance in terms of throughput, efficiency, cell activity, and space. The review explores the applications of single-cell isolation chips in genomics, epigenomics, transcriptomics, proteomics, and exosome analysis. It highlights the advantages and limitations of different chip technologies in these areas, emphasizing the importance of throughput, efficiency, and cell activity. For example, microwell chips are suitable for genomics and epigenomics due to their high throughput and efficiency, while droplet chips are powerful for large-scale DNA analysis in proteomics and exosome studies. The review concludes by discussing the current challenges and future prospects of microfluidic chip technology in single-cell analysis. It emphasizes the need for further advancements in cell activity, throughput, and efficiency to meet the demands of modern biological research. The integration of microfluidic chips with other technologies, such as nanopore sequencing, is also highlighted as a promising direction for future research.This review provides a comprehensive guide to researchers on the selection and application of microfluidic chip technologies for single-cell isolation and analysis. It highlights the design principles, separation mechanisms, chip characteristics, and cellular effects of various microfluidic chips, emphasizing their implications for subsequent multiomics and exosome analyses. The review also discusses the current challenges and future prospects of microfluidic chip technology in multiplex single-cell isolation and multiomic and exosome analyses. Single-cell multiomic and exosome analyses are crucial tools in various fields, including cancer research, immunology, neuroscience, microbiology, and drug development. Single-cell isolation is essential for reliable cell analysis, and microfluidic chips offer efficient and high-throughput solutions. The review covers the advantages and limitations of traditional single-cell isolation techniques, such as limiting dilution, laser capture microdissection (LCM), micromanipulation, and fluorescence-activated cell sorting (FACS), and introduces the benefits of microfluidic chips. The review discusses four main types of microfluidic chips for single-cell isolation: high-activity culture and multi-factor detection microchamber chips, high-efficiency and low-throughput double-layer valve chips, high-throughput and high-efficiency microdroplet chips, and high-throughput and high-efficiency microwell chips. Each type is evaluated based on its working principles, separation mechanisms, structural characteristics, and performance in terms of throughput, efficiency, cell activity, and space. The review explores the applications of single-cell isolation chips in genomics, epigenomics, transcriptomics, proteomics, and exosome analysis. It highlights the advantages and limitations of different chip technologies in these areas, emphasizing the importance of throughput, efficiency, and cell activity. For example, microwell chips are suitable for genomics and epigenomics due to their high throughput and efficiency, while droplet chips are powerful for large-scale DNA analysis in proteomics and exosome studies. The review concludes by discussing the current challenges and future prospects of microfluidic chip technology in single-cell analysis. It emphasizes the need for further advancements in cell activity, throughput, and efficiency to meet the demands of modern biological research. The integration of microfluidic chips with other technologies, such as nanopore sequencing, is also highlighted as a promising direction for future research.