The addition of simple substrates could affect the microbial respiration in soils. This substrate-induced respiration is widely used to estimate the soil microbial biomass, but little attention has been paid to its influence on the changes of community-level physiological profiles. In this study, the process of microbial communities responding to the added substrate using sole-carbon-source utilization (BIOLOG) was investigated. BIOLOG is biased toward fast-growing bacteria; this advantage was taken to detect the prompt response of the active microbial communities to the added substrate. Four soil samples from agricultural fields adjacent to heavy metal mines were amended with L-arginine, citric acid, or D-glucose. Substrate amendments could, generally, not only increase the metabolic activity of the microbial communities, but also change the metabolic diverse patterns compared with no-substrate control. By tracking the process, it was found that the variance between substrate-induced treatment and control fluctuated greatly during the incubation course, and the influences of these three substrates were different. In addition, the application of these induced changes to discriminate soil microbial communities was tested. The distance among all samples was greatly increased, which further showed the functional variance among microbial communities in soils. This can be very useful in the discrimination of microbial communities even with high similarity.