The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA amplification, was effectively applied to detect Salmonella in artificially spiked pork. The specificity of the developed PSR was tested using 16 Salmonella and 15 non-Salmonella strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was 4 × 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful, and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76 commercial pork samples. Therefore the positive predictive value, negative predictive value and accuracy rate of the developed assay were 100%. Considering its rapidity, user-friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a promising tool for the food industry for the detection of Salmonella and prevention of Salmonella outbreaks and recalls.