Three methods of BIOLOG microplate analysis with BIOLOG ECO Plate were applied and compared to explore the optimal way to analyze the carbon metabolism of farmland soils with carbon abundance. The soil samples were collected from Jingzhou Municipality and Huanggang Municipality,Hubei,China. The 100-fold eluted supernatant was incubated after incubating on ice. The 1 000-fold eluted suspension was incubated after incubating on ice. The supernatant was centrifuged after removing carbon. Color development of each well with optical density at 595 nm and 750 nm every 12 h was recorded with an ELISA Reader. The microbial activity in each microplate was expressed as average well color development (AWCD). We found that incubating 100-fold eluted supernatant could not accurately reflect the microorganism activities due to the available carbon in the supernatant which caused the reaction in the control well. The other two methods can clean up the carbon. The standard of AWCD of the method of incubating 1 000-fold eluted suspension after incubating on ice was significantly higher than that of incubating the supernatant after removing carbon with centrifuging,indicating that the latter one was more stably to detect the carbon metabolism of farmland soils.