Abstract: Variable domain of heavy chain antibody （VHH） is currently the smallest natural antibody molecule fragment with complete function which has a broad application prospect in the fields of basic research and drug development. The aim of the present study was to construct a natural single domain antibody library of Bactrian camel and to preliminarily identify the established antibody library. Total RNA was extracted from peripheral blood lymphocytes of five non-immunized healthy Bactrian camels, then reversely transcribed to cDNA, and amplified by PCR to obtain the gene which encoding the camel antibody variable region. By ligation with the phage vector, the recombinant phage was electroporated into competent cells of E. coli TG1 to construct a natural single domain antibody library of Bactrian camel. The results demonstrated that the size of the constructed library was 4.4 × 107 and the transformation positive rate was 75%; the variations in sequence and length of CDR3 region indicated the good diversity of the constructed library. A natural single domain antibody library of Bactrian camel is successfully constructed in this study, which will be fundamental for the valuable single domain antibody screening for specific antigens.