The aim of this study was to evaluate the values of immunophenotype of leukemic cells in bone marrow (BM) or peripheral blood (PB), lymphocytic subsets and NK cells of PB in diagnosis, therapy and prognosis of patients with B cell chronic lymphoid leukemia (B-CLL). The samples of PB and BM from 67 patients with B-CLL were collected and detected by multi-parameter flow cytometry. The results showed that the percentage of lymphocytes in PB of 67 patients with B-CLL obviously increased, as compared with normal persons, while CD3, CD4, CD8 and NK cells decreased significantly (p < 0.01), and no CD4/CD8 ratio had notable change (p > 0.05). In their immunophenotype results, the positive expression rate of CD19 was highest (91.04%), followed by CD5 (80.60%), CD 20 (76.12), cyCD 79a (74.63%), CD 38 (43.28%), CD 11c (42.86%), CD 7 (41.94%), ZAP-70 (39.29%), CD25 (0.00%); co-expression rate of CD5 and CD19 was 72.73%; the expression of ZAP-70 was correlated with CD 38 (p < 0.05). In conclusion, detection of cell immunophenotypes, lymphocytic subsets in PB and BM and NK cells in PB contribute to diagnosis of patients with B-CLL.