In this study, a real-time reverse transcriptase PCR (real-time RT-PCR) assay for the detection of Salmonella was designed and developed. The real-time RT-PCR assay targeted the multi-copy RNA, tmRNA, had an inclusivity and exclusivity of 100% and a sensitivity of ≤ 1 cell equivalent. The assay was combined with culture enrichment and an initial validation was performed in accordance with ISO 16140: 2003. Culture enrichment required 18 h primary enrichment in buffered peptone water (BPW) and 6 h selective enrichment in Rappaport–Vassiliadis Soya Peptone Broth (RVS). The combined culture enrichment/real-time RT-PCR method had a relative specificity of 100% when compared to the traditional culture method. When compared to an assay targeting the ssrA gene, which encodes for tmRNA, an approximate 1000-fold increase in sensitivity of detection was observed. Targeting the multi-copy tmRNA transcript rather than its encoding gene improves assay sensitivity which should enable reduction in turn-around time of this alternative method for Salmonella testing.