Photodynamic diagnosis is used during glioma surgery. Although some studies have shown that the spectrum of fluorescence was efficient for precise tumor diagnosis, previous methods to characterize the spectrum have been problematic, which can lead to misdiagnosis. In this paper, we introduce a comparison technique to characterize spectrum from pathology and results of preliminary measurement using human brain tissues. We developed a spectrum scanning system that enables spectra measurement of raw tissues. Because tissue preparations retain the shape of the device holder, spectra can be compared precisely with pathological examination. As a preliminary analysis, we measured 13 sample tissues from five patients with brain tumors. The technique enabled us to measure spectra and compare them with pathological results. Some tissues exhibited a good relationship between spectra and pathological results. Although there were some false positive and false negative cases, false positive tissue had different spectra in which intensity of short-wavelength side was also high. The proposed technique provides an accurate comparison of quantitative fluorescence spectra with pathological results. We found that spectrum analysis may reduce false positive errors. These results will increase the accuracy of tumor tissue identification.