Sydney Harbour is a global hotspot for marine and estuarine diversity. Despite its social, economic and biological value, the available knowledge has not previously been reviewed or synthesised. We systematically reviewed the published literature and consulted experts to establish our current understanding of the Harbour’s natural systems, identify knowledge gaps, and compare Sydney Harbour to other major estuaries worldwide. Of the 110 studies in our review, 81 focussed on ecology or biology, six on the chemistry, 10 on geology and 11 on oceanography. Subtidal rocky reef habitats were the most studied, with a focus on habitat forming macroalgae. In total 586 fish species have been recorded from the Harbour, which is high relative to other major estuaries worldwide. There has been a lack of process studies, and an almost complete absence of substantial time series that constrains our capacity to identify trends, environmental thresholds or major drivers of biotic interactions. We also highlight a lack of knowledge on the ecological functioning of Sydney Harbour, including studies on microbial communities. A sound understanding of the complexity, connectivity and dynamics underlying ecosystem functioning will allow further advances in management for the Harbour and for similarly modified estuaries around the world.