In this study, we have demonstrated the initial concept of fluorescence-polarization microscopy for surgical margin assessments. In future studies, the fluorescence and polarization signals could be calibrated to generate standard chart for quantitative measurement of cancer invasion, facilitating cancer staging. Similarly, many other pathological examinations can be explored with this new technique. Compared to other emerging histological techniques, such as confocal microscopy, two-photon fluorescent microscopy and single harmonic generation, the method reported herein allows for wide-field real-time imaging within a large field of view, which is the key for rapid histopathological assessment of tissue samples. Although the current setup is designed and tested in ex vivo settings, a similar concept can be extended to intravital and dissection microscopy. Although we used the tumor-targeted NIR molecular probe, LS301, for the fluorescent contrast, other cancer-targeting imaging agents may be also used in future studies. For example, molecular probes with diverse targeting mechanisms and spectra characteristics may be used to augment the utility of fluorescence microscopy in the clinical settings. The real-time feature of polarization contrast provides cross-validation and structural reference for fluorescence contrast.