<p>The completion of the Human Genome Project catalyzed the development of ‘omics’ technologies, enabling the detailed exploration of biological systems at an unprecedented molecular scale. Microfluidics has transformed the omics toolbox by facilitating large-scale, high-throughput and highly accurate measurements of DNA and RNA, driving the transition from bulk to single-cell analyses. This transition has ushered in a new era, moving beyond a gene- and protein-centric perspective toward a holistic understanding of cellular phenotypes. This emerging ‘single-cell phenomics era’ integrates diverse omics datasets with spatial, morphological and temporal phenotypes to provide a comprehensive perspective on cellular function. This Review highlights how microfluidics addressed key challenges in the transition to single-cell omics and explores how lessons learned from these efforts will propel the single-cell phenomics revolution. Furthermore, we discuss emerging opportunities in which integrative single-cell phenomics could serve as a foundation for transformative discoveries in biology.</p>