By using two-photon excited fluorescence (TPEF) and second harmonics generation (SHG), we imaged fresh ex vivo thyroid tissues, including normal thyroid tissue,nodular thyroid tissue and papillary thyroid carcinoma tissue. As can be seen, the follicles in normal thyroid tissue are in uniform size and shape, while in nodular thyroid tissue the follicles are in different size, and there are abundant substantial cancer cells in papillary thyroid carcinoma. These results are consistent with the corresponding standard H&E histology imagings. Furthermore, the collagen distributions in normal thyroid tissue and nodular thyroid tissue are observed to be different. These results suggest that the two-photon fluorescence microscopy imaging can distinguish, the morphologic differences between normal thyroid tissue,nodular thyroid tissue and papillary thyroid carcinoma tissue in microstructure, and it has the potential to be used for minimal invasive and rapid diagnosis of thyroid cancer.