Objective To construct phage display antibody library of artificial mutation to compare with the sequence of the natural phage display antibody library. To scientifically evaluate the quality of the artificial mutation of phage display library, and provide some references for the further transformation of the nanobody. Methods Using random mutation method, NNY fixed-point santuration mutation was performed on combine the follicle-stimulating hormone receptor (FSHR) of human nanobody. The mutant DNA sequence was connected to the vector pMECS to construct the phage display library of VHH06-CDR3 random mutation. By sequencing and analysis of DNA sequences, the diversity of the library and the amino acid distribution of CDR3 were compared between mutation library and the immune library of FSHR. The degree of enrichment of cloning was determined by six rounds of affinity screening. Results According to the NNY mutation rule ,the CDR3 regions with 16 amino acids by random mutations was synthesized and the VHH-CDR3 random mutant phage display library was constructed . The phage display library of VHH06-CDR3 random mutant size was 7.36×108 cfu/ml. Polyclonal and monoclonal phage ELISA showed that after six rounds of screening, the output phage and the combination of FSHR showed obvious enrichment, but there was no clone combined with FSHR. Conclusions Although the VHH06-CDR3 mutant phage display library has sequence diversity, it is not conducive to obtaining target antibodies in affinity screening due to the lack of functional diversity of CDR3.