Microbes can show sensitive response to environmental change via the variation of cell membrane lipids. This enables the proposal and establishment of a series of molecular proxies for paleoenvionmental conditions. Among them,U_(37)~k of algal unsaturated long-chained ketones,TEX_(86) of archaeal tetraethers,and the MBT /CBT of bacterial tetraethers are the most widely-used proxies in paleoenvironmental reconstruction. However,the proxies were found difficult in practical application in some cases,which awaits the systematic analysis on the microbial physiology and ecology. Methods: Molecular data of a variety of proxies were compiled from different microbes in different regions with various environmental conditions in modern soils in the world. Comparison was made among the molecular data published,which enables us to investigate the biogeochemical,physiological,and ecological differences in microbial responses to the ambient environmental conditions. Results: Three aspects related to the application of proxies were proposed to be deciphered,including the cross-check of multiple proxies due to the various biogeochemical processes,the sensitivity and the linear range of the proxies resulting from physiological responses,and the spatial heterogeneity of the proxies arising from the ecological difference. Biogeochemically,microbes can show responses to the ambient environmental conditions via several molecular patterns and pathways,providing a unique chance for the cross-check of multiple proxies. Physiologically,microbes are found to show a linear response to the environmental conditions only in certain range. The linear physiological response thus determines the sensitivity of the proxies for paleoenvironmental reconstruction. Ecologically,microbes might show responses to different environmental factors in different regions. The heterogeneity of ecological responses could affect the proxies used in different regions. Microbial activities in a specific environment might be restricted by a specific factor,such as the aridity in arid region,which in turn leads to the molecular proxies in a sensitive response to this particular factor instead of other factors. Conclusions: Application of microbial molecular proxies awaits the systematic investigations on all the biogeochemical,physiological and ecological responses. Care should be taken when we use these proxies without any knowledge of physiology and ecology of the corresponding microbes.