Microfluidic biochips are crucial for single-cell isolation and analysis of multiomics and exosomes, offering efficient, high-throughput, and real-time capabilities. These chips enable reliable cell isolation and quality control, essential for downstream analyses. This review discusses various microfluidic chip technologies for single-cell isolation, their design principles, separation mechanisms, and impacts on cellular functions, particularly in multiomics and exosome analysis. It also outlines current challenges and future prospects. Traditional single-cell isolation techniques, such as limiting dilution, laser capture microdissection, micromanipulation, and FACS, have limitations in efficiency, throughput, and cell activity. Microfluidic chips, with their small size and ability to handle small volumes, offer advantages in sensitivity and specificity. They enable high-throughput, high-efficiency, and high-activity single-cell isolation, which is critical for multiomics and exosome analysis. Several microfluidic chip technologies are discussed, including microchamber chips, double-layer valve chips, microdroplet chips, and microwell chips. Each has unique advantages and limitations. Microchamber chips allow high-activity culture and multi-factor detection, while double-layer valve chips offer high-efficiency and low-throughput isolation. Microdroplet chips provide high-throughput and high-efficiency isolation, but face challenges in cell activity and spatial indicators. Microwell chips enable high-throughput and high-efficiency isolation, but may affect cell growth due to small spaces. These technologies are applied in various fields, including genomics, epigenomics, transcriptomics, proteomics, and exosome analysis. Genomics and epigenomics often require fixed, permeabilized, or lysed cells, while proteomics and exosome analysis require high-activity single-cell isolation. Microfluidic chips are particularly suitable for electrical signal analysis due to their ability to maintain cell activity. The review highlights the importance of microfluidic chip technology in advancing single-cell analysis, emphasizing the need for high-throughput, high-efficiency, and high-activity isolation. It also discusses the challenges and future directions for improving these technologies to meet the demands of multiomics and exosome analysis.Microfluidic biochips are crucial for single-cell isolation and analysis of multiomics and exosomes, offering efficient, high-throughput, and real-time capabilities. These chips enable reliable cell isolation and quality control, essential for downstream analyses. This review discusses various microfluidic chip technologies for single-cell isolation, their design principles, separation mechanisms, and impacts on cellular functions, particularly in multiomics and exosome analysis. It also outlines current challenges and future prospects. Traditional single-cell isolation techniques, such as limiting dilution, laser capture microdissection, micromanipulation, and FACS, have limitations in efficiency, throughput, and cell activity. Microfluidic chips, with their small size and ability to handle small volumes, offer advantages in sensitivity and specificity. They enable high-throughput, high-efficiency, and high-activity single-cell isolation, which is critical for multiomics and exosome analysis. Several microfluidic chip technologies are discussed, including microchamber chips, double-layer valve chips, microdroplet chips, and microwell chips. Each has unique advantages and limitations. Microchamber chips allow high-activity culture and multi-factor detection, while double-layer valve chips offer high-efficiency and low-throughput isolation. Microdroplet chips provide high-throughput and high-efficiency isolation, but face challenges in cell activity and spatial indicators. Microwell chips enable high-throughput and high-efficiency isolation, but may affect cell growth due to small spaces. These technologies are applied in various fields, including genomics, epigenomics, transcriptomics, proteomics, and exosome analysis. Genomics and epigenomics often require fixed, permeabilized, or lysed cells, while proteomics and exosome analysis require high-activity single-cell isolation. Microfluidic chips are particularly suitable for electrical signal analysis due to their ability to maintain cell activity. The review highlights the importance of microfluidic chip technology in advancing single-cell analysis, emphasizing the need for high-throughput, high-efficiency, and high-activity isolation. It also discusses the challenges and future directions for improving these technologies to meet the demands of multiomics and exosome analysis.