Single-cell metabolomics can be used to study cell diversity and how cells respond to environment. There is an urgent need to develop effective detection methods for single-cell metabolomics. Microchip electrophoresis with laser-induced fluorescence detection (MCE-LIFD) is a powerful tool to detect metabolites at the single-cell level. However, the existing one-laser excitation and one-color fluorescence collection in MCE-LIFD is not sufficient for the simultaneous detection of multiple small molecules with wide variations in their fluorescence excitation and emission spectra. In this manuscript, we describe a multicolor fluorescence detection-based microfluidic device (MFD-MD) for single-cell metabolomics research. We selected primary liver cells from acute ethanol-stimulated mice as the model cells and hydrogen peroxide (H2O2), glutathione (GSH), and cysteine (Cys) as representative small-molecule metabolites for single-cell analysis. The microfluidic chip enabled accurate single-cell manipulation and effective electrophoresis separation. The new multicolor fluorescence detection permitted simultaneous analysis of H2O2, GSH, and Cys. Ethanol exposure induced an increase in H2O2 and a decrease in GSH and Cys. Obvious cell heterogeneity was observed. These results provide insights regarding the intracellular oxidative/antioxidative molecular mechanism in response to external stimuli. The MFD-MD provides a new opportunity for simultaneous single-cell analysis of multiple metabolites.