A quantitative PCR method (qPCR) was developed for the detection and quantification of Ramularia beticola causing Ramularia leaf spot in sugar beet. R. beticola specific primers were designed based on the internal transcribed spacer region 2 (ITS2). The assay was applied on DNA extracted from spores trapped on tape from Burkard spore traps placed in an artificially inoculated sugar beet field trial and in two sugar beet fields with natural infections. R. beticola DNA was detected at variable amounts in the air samples 14 to 16 days prior to first visible symptoms. R. beticola DNA was detected in air samples from fields with natural infection at significant and increasing levels from development of the first symptoms, indicating that spore production within the crop plays a major role in the epidemic development of the disease. Sugar beet leaves sampled from the inoculated field trial were also tested with the qPCR assay. It was possible to detect the presence of R. beticola in the leaves pre-symptomatic at least 10 days before the occurrence of the visible symptoms of Ramularia leaf spot. This is the first report of a molecular assay, which allows screening for the presence of R. beticola in plant material and in air samples prior to the appearance of visible symptoms. An early detection has potential as a tool, which can be part of a warning system predicting the onset of the disease in the sugar beet crop and helping to optimise fungicide application.