Simultaneous acquiring chemical and structural information within the biofilm matrix is vital for the cognition and regulation of biofilms, yet it remains a great challenge due to the sample complexity and the limited approaches. Here, we combined confocal Raman microscopy with non-negative matrix factorization (NMF) analysis to explore the spatiotemporal organization of Escherichia coli biofilms, from the bacterial attachment to the biofilm maturation. Four components, including proteins and RNA, cells, lipopolysaccharides/lipoprotein and polyhydroxybutyrate were successfully resolved. Meanwhile, the corresponding 3D structure could be visualized and quantified by their abundances derived from the NMF analysis. This approach reveals an intimate relationship between the spatiotemporal distribution and the biomolecular specificity and suggest a promising perspective for understanding the mechanism of biofilm development at molecular-level detail.