The present invention belongs to field of biology and medicine, and especially relates to a novel antibiotic comprising an antibody mimetic antibody, its preparation methods and uses thereof. A novel antibiotic comprising a antibody mimetic covalently bonded to the carboxyl end of a colicin polypeptide or a channel-forming domain polypeptide of a colicin, wherein said colicin is selected from the group consisting of Colicin E1, Ia, Ib, A, B, N wherein said antibody mimetic being yielded by fusing two complementarity determining regions (CDRs), VHCDR1 and VLCDR3 through a cognate framework region (VHFR2) of an immunoglobulin wherein said the immunoglobulin specifically recognizes the bacterial porins. Its antibacterial ability is a thousandfold powerful than normal antibiotics. Due to its unique action mechanism, drug resistance resulted in mutation can hardly be acquired by pathogenic bacteria. And the antibiotic will not hurt normal human cells when it kills pathogenic bacteria. Therefore, it can be used for manufacturing antibacterial medicament of killing Neisseria meningitidis, vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, multidrug-resistance Pseudomonas aeruginosa or Mycobacterium tuberculosis.
