Seamless modification of bacteria chromosome is widely performed both in theoretical and in practical research, for this purpose, excellent counter-selection marker genes with high selection stringency are needed. Lysis gene E from bacteriophage PhiX174 was developed and optimized as a counter-selection marker in this paper. Lysis gene E was firstly constructed under the control of pL promoter. At 42 °C, Lysis gene E could effectively kill Escherichia coli. Seamless modification using E as a counter-selection marker also successfully conducted. It also works in another Gram-negative strain Serratia marcescens under the control of Arac/PBAD regulatory system. Through combining lysis gene E and kil, the selection stringency frequency of pL-kil-sd-E cassette in E. coli arrived at 4.9×10−8 and 3.2×10−8 at two test loci, which is very close to the best counter-selection system, inducible toxins system. Under the control of Arac/PBAD, selection stringency of PBAD-kil-sd-E in S. marcescens arrived at the level of 10−7 at four test loci. By introducing araC gene harboring plasmid pKDsg-ack, 5- to 18- fold improvement of selection stringency was observed at these loci, and a surprising low selection stringency frequency 4.9×10−9 was obtained at marR-1 locus, the lowest selection stringency frequency for counter-selection reported so far. Similarly, at araB locus of E. coli selection stringency frequency of PBAD-kil-sd-E was improved to 3×10−9 after introducing plasmid pKDsg-ack. In conclusion, we have developed and optimized a newly universal counter-selection marker based on lysis gene E. The best selection stringency of this new marker exceeds the inducible toxins system several fold.