We formulated a selective enrichment broth (SVV) for simultaneous growth of Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae by single factor experiment and response surface method. We evaluated the enrichment effect of SVV by conventional culture method and real-time PCR assay. We obtained the SVV broth by supplementting the Buffered Peptone Water (BPW) with bile salt no. 3, potassium tellurite, and sodium citrate as inhibitors, and glucose, mannitol, snhydrous sodium sulfite and sodium pyruvate as accelerants. We also modified the concentration of sodium chloride in BPW. When mixed at equal or varied proportions, the target pathogens had a great accumulation (10(5)-10(8) CFU/mL) after incubated in SVV for 18 h at 37 degrees C with shaking. It can also effectively inhibit the competitive microflora. We detected 10 artificial simulated samples and 608 real samples using SVV with real-time PCR. After enriched in SVV for 18 h, the quantity of the bacteria in samples were above the detection limit. The SVV with PCR assay showed higher tested positive (4.06%) compared to that of the conventional detection method (3.78%) and there was no false report. In summary, SVV is a promising new multiplex selective enrichment broth that can be used in detection of seafood.