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In this study, the spatial distribution and accumulation dynamics of volatile oil in Angelica sinensis roots was realized by fluorescence imaging combined with mass spectrometry imaging. The laser scanning confocal microscopy was used to determine the optimal excitation wavelength and the fluorescent stability of volatile oil in the sections of Angelica sinensis roots. The results demonstrated that 488 nm was the most suitable excitation wavelength for the identification and quantitative analysis of volatile oil. It was observed that volatile oil accumulated in the oil chamber of the phelloderm and secondary phloem, and the oil canal of the secondary xylem. The results also indicated that there were differences in content during different periods. Furthermore, the MALDI-TOF-MSI technology was used to study the spatial distribution and compare the chemical compositions of different parts of Angelica sinensis roots during the harvest period. A total of 55, 49, 50 and 30 compounds were identified from the head, body, tail of the root and root bark, respectively. The spatial distribution of phthalides, organic acids and other compounds were revealed in Angelica sinensis roots. The method developed in this study could be used for the in situ analysis of volatile oil in Angelica sinensis roots.