Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria are important pathogens for human. With increased awareness in public health, development of a rapid, sensitive, cost-effective and easy-operating bacteriological detection is of the utmost importance and urgent necessity. In this study, we developed and applied a simple amplification kit based on loop-mediated isothermal amplification (LAMP) methods for rapid detection of various pathogens including Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria, as well as related virulence. Nine targets, including rfbE (E. coli-specific), stx1 (coding for Shiga toxin 1), stx2 (coding for Shiga toxin 2), oprI (P. aeruginosa–specific), invA (Salmonella-specific), hlyA (Listeria-specific), tlh (coding for thermolable haemolysin), tdh (coding for thermostable direct haemolysin) and trh (coding for TDH-related haemolysin), were selected for identification for Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria, as well as related virulence.. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on the targets. Three solutions labeled A, B and C was included in the kit. The experiment were carried out in a total of 25 μl reaction mixture: solution A containing 1.6 μM (each) of the primers FIP and BIP, 0.2 μM (each) of the primers F3 and B3, 0.8 μM (each) of primers LF and LB; solution B containing 1.6 mM of deoxynucleoside triphosphates, 6 mM MgSO4, 1 M betain (Sigma, St. Louis, MO, USA), 1 X thermopol buffer (New England Biolabs, Ipswich, MA, USA); solution C containing BST polymerase. Twenty-two reference strains, including various species of gram-negative and-positive isolates, were included in this study to evaluate and optimize LAMP assays. Application of the optimized LAMP assays was performed on a total of 200 strains (with 40 strains for each species of the pahtogens). The optimal reaction condition was found to be 65°C for 45 min. Application of the kit assays were performed on various types of pathogens, the sensitivity for the 9 targets was found to be 100%; with a 100% specificity and positive predictive value (PPV) for all the 9 targets targets. In conclusion, the isothermal amplification kits were demonstrated to be useful and powerful tools for rapid differentiation of various pathogens (including Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria), and undoubtedly, the rapidness, easiness and cost-effectiveness of LAMP assay will aid in the broad application of bacteriological detection of common pathogens.