Background Peripherally inserted central catheters (PICCs) are susceptible to Staphylococcus aureus (S. aureus) colonisation and subsequent dissemination into the bloodstream, leading to central line-associated bloodstream infections (CLABSI). Current detection for S. aureus PICC colonisation relies on the use of traditional culture-dependent methods, including the semi-quantitative roll-plate culture method. However, the minimum time to detection is between 24–48 hours. Furthermore, a definitive diagnosis may take up to 7 days and is therefore not useful in guiding appropriate and timely patient management. A quantitative real-time polymerase chain reaction (qPCR) assay has the potential to overcome these limitations. Methods A qPCR assay, targeting the nuclease (nuc) gene, was developed to detect S. aureus PICC colonisation. The sensitivity threshold of the assay was determined using purified S. aureus genomic DNA (gDNA) and validated using 41 clinical PICC samples which were compared to results from the roll-plate culture method. Results The sensitivity threshold of the qPCR assay was 102 CFU/mL-1. From a total of 41 clinical PICC samples, S. aureus colonisation was detected from one PICC by both qPCR (103 CFU/mL-1) and the roll-plate culture method (103 CFU/mL-1). The qPCR assay processing time was less than 2 hours after bacterial gDNA isolation compared with 24–48 hours for the roll-plate culture method. Conclusion This developed qPCR assay is an accurate and rapid method to detect S. aureus PICC colonisation. With further research, this method has the potential to be used in a clinical setting.