Foodborne pathogens can cause foodborne illness. In reality, one food sample may carry more than one pathogen. A rapid, sensitive, and multiple target method for bacteria detection is crucial in food safety. For the simultaneous detection of Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella Enteritidis, multi-objective recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) was developed in this study. The whole process, including amplification and reading, can be completed in 15 min at 37 degrees C. The detection limits were 2.6 x 10(1) CFU/mL for Staphylococcus aureus, 7.6 x 10(1) CFU/mL for Vibrio parahaemolyticus, and 1.29 x 10(1) CFU/mL for Salmonella Enteritidis. Moreover, colored signal intensities on test lines were measured by a test strip reader to achieve quantitative detection for Staphylococcus aureus (R-2 = 0.9903), Vibrio parahaemolyticus (R-2 = 0.9928), and Salmonella Enteritidis (R-2 = 0.9945). In addition, the method demonstrated good recoveries (92.00%-107.95%) in the testing of spiked food samples. Therefore, the multiplex LFD-RPA assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens in seafood
