Abstract Background We compared the cell counting accuracy of the conventional method and the improved method by using Neubauer counting chamber. Methods In the improved method, all the border cells were counted and then divided by two; while, in the conventional method, only border cells on the two boundaries (top and left) were counted. Results About 55.814% of the samples showed more accurate results by improved counting method, about 38.372% had more accurate results by conventional counting method, and about 5.814% were counted with similar counting error by both methods. The improved method had significantly smaller counting error than conventional method ( P < .05). The distribution ratio of the border cells was an independent factor for counting accuracy ( P < .05). Conclusion Together, the improved counting method can reduce the counting error of the Neubauer counting chamber to some extent, assess the distributing uniformity of border cells, and help to eliminate the samples with large differences in distribution.