Objective: To establish a method cloning the exon 3 of eck gene from normal tissue and ZR-75-1 cell line (a human breast cancer cell line) and study whether these genes exist mutant. Methods: Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR-75-1 cell line respectively by PCR technique. Transformed the E. coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm-T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results: (1) Obtained the genomic DNA of human normal epithelial cells and ZR-75-1 cell line respectively. (2) Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. (3) Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR-75-1 cell line respectively. (4) ZR-75-1 cell line exists mutation of nucleotides. Conclusion: Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.