Background The complete blood count (CBC) is a basic test routinely ordered by physicians as a part of initial diagnostic work-up on their patients. To ensure safe clinical application of the CBC, reliable biological variation (BV) data are needed to establish analytical performance specifications. Our aim was to define the BV of CBC parameters using a rigorous protocol that is compliant with the Biological Variation Data Critical Appraisal Checklist (BIVAC) provided by the European Federation of Clinical Chemistry and Laboratory Medicine. Methods Blood samples drawn from 41 healthy Chinese subjects (22 females and 19 males; 23-59 years of age) once monthly for 6 consecutive months were analyzed using an ABX Pentra 80 instrument. The instrument was precisely calibrated. All samples were analyzed in duplicate for 13 CBC parameters. The data were assessed for outliers, normality, and variance homogeneity prior to nested ANOVA. Gender-stratified within-subject (CVI) and between-subject (CVG) BV estimates were calculated. Results The number of remaining data for each subject was 442-484 after removing outliers. No significant differences existed between female/male CVI estimates. Except for leukocytes, neutrophils, and lymphocytes, the mean values of 10 parameters differed significantly between genders, rendering partitioning of CVG data between genders. No significant differences were detected between most BV estimates and recently published estimates representing a Europid population. Conclusions Most BV estimates in BIVAC-compliant studies are similar. The turnover time of blood cells and age distribution of participants should be considered in a CBC BV study. Our study will contribute to global BV estimates and future studies.