Spruce is the commonly-used tonewood for the top plate of violin-family instruments, such as violins and cellos. The wood properties can critically determine the acoustic quality. It’s been shown the wood of famous old instruments differ from modern ones due to chemical treatment and aging. To reveal the differences microscopically in both spatial and spectral domains, a two-photon hyperspectral system has been applied to investigate the autofluorescence and second harmonic generation within wood samples. Not only the cellular structures were observed through optical sectioning, but the spectral variations were revealed among different age wood samples and different cellular structures.