Objective The pulsed field gel electrophoresis (PFGE) typing method of Aeromonas was optimized from aspects of restriction enzymes and electrophoresis parameters, which reduced fragments co-migration and made them well-distributed for easily analyzing. Methods Using whole genome sequences of 6 Aeromonas, restriction fragments produced by 677 enzymes were analyzed in BioEdit software. Enzymes producing more effective fragments in appropriate number were selected, and the fitting curve and regression equation were derived from the length of restriction fragments represented as denary logarithm and the relative position of fragments in gel represented as fragments migration distance percentage of the valid length of the reference system. Digital simulation of pulsed field gel electrophoresis map was shown when length of fragments from different Aeromonas substituted into the equation. Results of digital simulation were also verified by PFGE typing assay with selected enzymes for Aeromonas typing. Isolates from diarrhea patients were subtyped by the optimized new method. Results BioEdit analysis result showed PmeⅠ, PacⅠand SwaⅠ were better than XbaⅠ as enzymes for PFGE from the aspects of total and effective number of restriction fragments. According to the result of PFGE simulation and the verification test of isolates, the distribution of fragments of PacⅠ and PmeⅠ was better distributed and less overlapped than those of XbaⅠ and SwaⅠ. Finally, PmeⅠ and PacⅠ were selected as the first and second choice of enzymes for PFGE typing, and the pulsed field conversion time was set as 2.16-63.8 s. Sixteen isolates from diarrhea patients were subtyped by new method, and the Simpson diversity index was 99.17% according to the typing result. Conclusion As enzymes used in PFGE typing, PmeⅠ and PacⅠ were better choices than XbaⅠ, and good resolution was obtained from subtyping of isolates from diarrhea. The procedure for optimizing PFGE typing method provided in this study is applicable to the establishment or optimization of other bacteria's PFGE typing method.