AIM: To construct a CS3 fimbria-displayed random peptide library on the Escherichia coli cell surface. METHODS: Firstly, a new display vector with double restriction sites was reconstructed, and the ability of the new vector to form CS3 fim- briae on E. coli surface was confirmed by West - ern blot and transmission electron microscopy. Then two oligonucleotides were synthesized, one of which contained a random oligonucle- otide encoding region (NNK)10 and the other was its complement. The two synthesized oligo- nucleotides was annealed and then extended by Klenow Fragment. The double-stranded oligo- nucleotides were digested by XhoⅠ and BamH Ⅰ and purified by PAGE, then inserted into the digested display vector. The ligation product was purified and electroporated into XL1-Blue. Ten randomly selected clones were sequenced and the sequences were analyzed. RESULTS: A library with diversity of 1.8×10 6