Abstract: As one of the main methods of microbial community functional diversity measurement, biolog method is favored by many researchers for its simple operation, high sensitivity, strong resolution and rich data. But the preprocessing methods reported in the literature are not the same. In order to screen the best preprocessing methods, this paper took three typical treatments to explore the effect of different preprocessing methods on soil microbial community functional diversity. The results showed that, method B’s overall trend of AWCD values was better than A and C’s. Method B’s microbial utilization of six carbon sources was higher, and the result was relatively stable. The Simpson index, Shannon richness index and Carbon source utilization richness index of the two treatments were B > C > A, while the McIntosh index, Shannon evenness was not very stable, but the difference of variance analysis was not significant, and the method B was always with a smallest variance. Method B’s principal component analysis was better than the A and C. In a word, the method using 250 r/min shaking for 30 minutes and cultivating at 28℃ was the best one, because it was simple, convenient, and with good repeatability.