The concentration, spatial distribution and speciation of elements in environmental matrices are the key to understanding their biological functions and environmental behaviors. This paper aims to review the recent applications and challenges of X-ray spectrometry in biology and ecological environment. It is demonstrated that X-ray fluorescence spectroscopy analysis can provide quantitative data on the translocation and distribution of elements in living plants. Micro-X-ray fluorescence and X-ray absorption spectroscopy are unique in providing in situ information, and both help to understand the interaction between organisms and elements, especially the uptake, transport, accumulation and detoxification mechanism of elements in organisms. Meanwhile, both are also used to reveal the environmental behaviors such as the source, evolution and fate of elements in typical environmental samples. However, due to the complexity and diversity of biological and environmental matrices, there are still some technical difficulties and challenges, such as overcoming the self-absorption effect of X-ray fluorescence, accurately identifying the low-abundance (5%~10%) elemental species, and rapidly arresting the transient redox reactions of elements in living cells.