Background: The aim of this study was to prove the difference between 37 degrees C water incubation method and the BEP III automated immunoassay analyzer incubation method in anti-EBNAl-IgA antibody detection and to find the best way to improve the consistency of the two incubation methods. Methods: The 37 degrees C water incubation method and BEP III analyzer incubation method were used with the same panel of samples (n = 39) in anti-EBNAl-IgA antibody detection. Except for incubation, the rest of the steps were performed by the BEP III analyzer in both groups. All the data were analyzed by SPSS 17.0 software. Line charts and bar charts were used to compare the difference between the two incubation methods in anti-EBNAl-IgA antibody detection. We planned to find the best incubation scheme for BEP III analyzer, consistent with the water incubation method, using three groups of prolonged incubation time experiments. Results: A sample panel of 39 outpatients were analyzed by two incubation methods. The results showed by line charts that the water incubation method had higher S/CO values than the BEP III analyzer incubation method. Meanwhile the water incubation group had more positive results (61.5%) and less borderline positive results (35.9%) than that of the BEP III analyzer incubation group which were 43.5% and 51.2%, respectively, in the stacked bar charts. We found that by prolonging the incubation time in the BEP III analyzer for 6 minutes in the first and second incubation steps the S/CO values we increased and achieved statistically coincident results with water incubation group. Conclusions: There were biases between the 37 degrees C water incubation method and the BEP III analyzer incubation method in anti-EBNAl-IgA antibody detection. The water incubation method had higher S/CO values than the BEP III analyzer incubation method in paired groups and led partly to a difference in test results. By prolonging the BEP III analyzer incubation time properly, it can reduce the difference to some extent resulting in statistically similar results with the water incubation method.