Objective: To construct, reproduce and identify the global microRNA-34a (miR-34a) gene knockout DBA1/J mice. Methods: According to the principle of CRISPR/cas9, miR-34a-/+DBA1/J mice were constructed as F0 generation mice; they were hybridized with DBA1/J wild type mice (WT), and miR-34a-/+F1 mice with genotype were screened out by PCR identification; miR-34a-/-F2 generation mice were screened out, and their growth characteristics and reproductive ability were analyzed; the proportion of T and B lymphocytes in immune organs and peripheral blood of mice was detected by flow cytometry. Results: Two F0 mice, four F1 mice and five F2 mice were obtained. The proportions of CD4+T, CD8+T lymphocyte subpopulations had no significant changes(P>0.05), but increased in the propotion of B lymphocytes in immune organs and peripheral blood of miR-34a-/- knockout mice(P<0.05). There were no significant differences in weights and reproductive capacities between the miR-34a knockout mice and WT DBA1/J mice(P>0.05). Conclusion: The global miR-34a knockout mice were constructed successfully via CRISPR/Cas9 technique.