The analysis of in situ single cells in tissue is crucial for the study of tumors. However, simultaneous analysis of multiple biomolecules for single cells by fluorescence immunohistochemistry is challenging. Spontaneous Raman spectroscopy provides biochemical fingerprints of individual cells in a label-free and nondestructive manner, yet it is not well applied to single cells in tissue. Here, we develop an in situ single-cell spontaneous Raman spectroscopy (SRS) system that can localize a single cell in tissue with fluorescence imaging, while obtaining the single-cell Raman spectra for molecular analysis. Our system is verified by measuring the standard polystyrene beads of different sizes. Measurements of in situ single macrophages in clinical cancerous and para-cancerous tissues are then performed. By using discriminant analysis, it is shown that CD68+ macrophages in cancerous and para-cancerous tissues are classified with an accuracy of 98.3 %. The molecular content of single macrophages in tissue is analyzed by using multivariate curve resolution-alternating least squares method. We find that the lipid content of tumor-associated macrophages in cancerous tissues is higher while both the contents of protein and nucleic acid are lower than those in para-cancerous tissues, which is challenging to achieve by conventional immunohistochemistry. Our in situ single-cell SRS system has the capability for the obtaining of molecular information of a localized single cell in tissue, which may find potential applications for clinical cancer diagnosis. •An in situ single-cell spontaneous Raman spectroscopy (SRS) system is developed.•In situ single-cell SRS localizes a single cell in tissue, and obtains its Raman spectra for molecular analysis.•Single CD68+ macrophages in liver cancerous and para-cancerous tissues are classified.•Various molecular substances of single CD68+ macrophages in liver cancerous and para-cancerous tissues are analyzed.