Since the 1990s, recognition of urban biodiversity research has increased steadily. Knowledge of how ecological communities respond to urban pressures can assist in addressing global questions related to biodiversity. To assess the state of this research field in meeting this aim, we conducted a systematic review of the urban biodiversity literature published since 1990. We obtained data from 1209 studies that sampled ecological communities representing 12 taxonomic groups. While advances have been made in the field over the last 30 years, we found that urban biodiversity research has primarily been conducted in single cities within the Palearctic and Nearctic realms, within forest remnants and residential locations, and predominantly surveys plants and birds, with significant gaps in research within the Global South and little integration of multi-species and multi-trophic interactions. Sample sizes remain limited in spatial and temporal scope, but citizen science and remote sensing resources have broadened these efforts. Analytical approaches still rely on taxonomic diversity to describe urban plant and animal communities, with increasing numbers of integrated phylogenetic and trait-based analyses. Despite the implementation of nature-based solutions across the world’s cities, only 5% of studies link biodiversity to ecosystem function and services, pointing to substantial gaps in our understanding of such solutions. We advocate for future research that encompasses a greater diversity of taxonomic groups and urban systems, focusing on biodiversity hotspots. Implementing such research would enable researchers to move forward in an equitable and multidisciplinary way to tackle the complex issues facing global urban biodiversity.