This disclosure pertains to the technical field of antibodies, and discloses PD-L1 nanobodies, preparation methods and uses thereof; two primers are designed and synthesized; a sufficient amount of target products is amplified by PCR to carry out replacement enzyme digestion; target fragments are ligated to vectors, transformed, and screened to obtain clones; proteins are identified and expressed; antibody library is constructed; M13 phage display system is selected to display VHH antibody library, which consists of pMECS phagemid vector, E. coli TG1 and M13K07 helper phage. In the phagemid vector pMECS of this disclosure, the sequence before the Pst I restriction site is the coding sequence of the pelB secretion signal peptide and part of the amino acids in the first framework region of the antibody; the pelB signal peptide can guide the secretion of the subsequent polypeptide to the periplasmic cavity; Not I restriction site is followed by the coding sequence of HA and 6×His tag, which can be used for purification or detection of fusion protein.