With the development of microbial ecology techniques, it is possible to analyze the distribution and function of microorganisms simultaneously in complex ecosystems. To explore the application of FISH-NanoSIMS in environmental microbial ecology study, our study used the stable isotope labeled compounds 13C-C6H12O6, and 15N-NH4Cl as C and N sources for cultivating the pure culture (manganese oxidizing bacteria, Pseudomonas sp. QJX-1) and environmental samples (the shallow soil and anaerobic sludge). FISH-NanoSIMS was used to detect the distribution of microorganisms and relatively quantify secondary ions (12C-, 13C-, 12C(14)N-, 12C15N-) in cultivated samples, in order to explore the utilization of C and N isotopes sources by the pure culture and microorganisms in environment samples. The results showed that the contents of 13C and 5N in the area of bacteria were significantly greater than the natural abundance in all samples. It indicated that Pseudomonas sp. QJX-1 and some specific bacteria in environmental samples could metabolize 13C-C6H12O6 and 15N-NH4C1. Furthermore, this study revealed that for Pseudomonas sp. QJX-1, the manganese oxidation only occurred when the carbon and nitrogen were consumed to a low level. For environmental samples, the bacterial nitrification and denitrification were both observed in the shallow soil and anaerobic sludge. In a word, our study demonstrated that the combination of FISH and NanoSIMS could simultaneously examine microbial distribution and microbial metabolic activity in environmental samples, which will help us to obtain the eco-physiology information of microbial community.