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A single cell is the basic functional unit of life.All living organisms start from single cells.Single-cell analysis can circumvent the bottleneck in cultivation and cellular heterogeneity,and enables understanding the bacterial function and its ecological role in the in-situ microbial community.A single-cell Raman spectrum (SCRS) provides the intrinsic chemical ‘fingerprint’ of an individual cell and can be obtained noninvasively.However the key drawback that Raman signal is naturally weak hampers the development of high-throughput single-cell Raman technology.Here we optimized our Raman-activated Cell Sorter (RACS) by shortening its light path and employing a low noise and high sensitive EMCCD detector.We identified the correlation of the signal noise ratio (SNR) of SCRS with laser power and acquisition time,and reduced the acquisition time of a SCRS to the millisecond level.The acquisition time of SCRS can be reduced to 100ms for bacterial cells and to 10ms for larger cells such as Saccharomyces cerevisiae.The SCRS of cells labeled with stable isotope (13C or 15N),obtained with the acquisition time of 0.1s,showed clear shifts at certain Raman bands caused by the incorporation of heavy atoms.The SCRS was consistent with those obtained previously with over 30s acquisition time,demonstrating that our overall two-order-magnitude reduction of SCRS acquisition time.did not compromise signal quality.Furthermore,PC-CVA analysis result of SCRS of five oral bacterial species obtained with 0.5s acquisition time each indicated that SCRS can be used for phylogenetic analysis of different species at the resolution of a single-cell.Our results demonstrated a labelfree,rapid approach for identifying cells of different phenotypes or phylotypes,and paved the way for highthroughput isolation and culture of single-cells based on their SCRS.