Raman spectroscopy is a nondestructive, label-free, highly specific approach that provides the chemical information on materials. Thus, it is suitable to be used as an effective analytical tool to characterize biological samples. Here we introduce a novel method that uses artificial intelligence to analyze biological Raman spectra and identify the microbes at a single-cell level. The combination of a framework of convolutional neural network (ConvNet) and Raman spectroscopy allows the extraction of the Raman spectral features of a single microbial cell and then categorizes cells according to their spectral features. As the proof of concept, we measured Raman spectra of 14 microbial species at a single-cell level and constructed an optimal ConvNet model using the Raman data. The average accuracy of classification by ConvNet is 95.64 ± 5.46%. Meanwhile, we introduced an occlusion-based Raman spectra feature extraction to visualize the weights of Raman features for distinguishing different species.