Objective To evaluate the chemiluminescence (ECL) method for the determination of red cell folate concentration. Methods Evaluation of the methods includes repeated test, recovery test, interference test, linear test, methodology comparison and establishment of the reference range. Then, the red cell folate levels of 30 pregnant women who intake folic acid for more than 60 days were measured. Results The intra and inter CV of the method were 2.57% and 3.55%, respectively. The recovery of this assay was 98.0%.The assay was linear from 40.2-1174.82ng/mL, and it wouldn’t be interference by triglyceride (148mg/dL) or bilirubin (0.6mg/dL). The reference range of red cell folate was 111.33 -603.75ng/mL. The results determined with ECL method and RIA had no significant difference. The results were also positively correlated (r=0.998). The red cell folate levels in 30 pregnant women who intake folic acid for more than 60 days had significant differences compared with the results of control group. Conclusion ECL can be an accurately and rapid method for determination of red cell folate concentrations, and this method is suitable for clinical application.