Functional enrichment analysis is pivotal for interpreting high-throughput omics data in life science. It is crucial for this type of tool to use the latest annotation databases for as many organisms as possible. To meet these requirements, we present here an updated version of our popular Bioconductor package, clusterProfiler 4.0. This package has been enhanced considerably compared with its original version published 9 years ago. The new version provides a universal interface for functional enrichment analysis in thousands of organisms based on internally supported ontologies and pathways as well as annotation data provided by users or derived from online databases. It also extends the dplyr and ggplot2 packages to offer tidy interfaces for data operation and visualization. Other new features include gene set enrichment analysis and comparison of enrichment results from multiple gene lists. We anticipate that clusterProfiler 4.0 will be applied to a wide range of scenarios across diverse organisms.