Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins， however， can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside， severely restricting the development of lysins against Gram-negative bacteria. In this study， genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK)， a 10 cationic amino acid polypeptide (KRKRKRKRKR)， a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK)， and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa， 10aa， 15aa， Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids， Mix， can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa， 10aa， 15aa) also showed bactericidal activity against E. coli from the outside， with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively， improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli， providing a new practical method for the development of anti-Gram-negative bacterial lysins.