Ross River virus (RRV) infection is the most common notifiable vector-borne disease in Australia, with around 6000 cases annually. This study aimed to examine the relationship between climate variability and notified RRV infections in the Riverland region of South Australia in order to set up an early warning system for the disease in temperate-climate regions. Notified data of RRV infections were collected by the South Australian Department of Health. Climatic variables and monthly river flow were provided by the Australian Bureau of Meteorology and South Australian Department of Water, Land and Biodiversity Conservation over the period 1992–2004. Spearman correlation and time-series-adjusted Poisson regression analysis were performed. The results indicate that increases in monthly mean minimum and maximum temperatures, monthly total rainfall, monthly mean Southern Oscillation Index and monthly flow in the Murray River increase the likelihood, but an increase in monthly mean relative humidity decreases the likelihood, of disease transmission in the region, with different time-lag effects. This study demonstrates that a useful early warning system can be developed for local regions based on the statistical analysis of readily available climate data. These early warning systems can be utilized by local public health authorities to develop disease prevention and control activities.