Hepatocellular　carcinoma　(HCC)　is　the　most　common　primary　malignant　liver　tumor　worldwide,　with　its　development　and　progression　influenced　by　the　complex　tumor　microenvironment　(TME).　Tumor　heterogeneity　is　a　crucial　factor　in　determining　the　biological　behavior　and　malignancy　of　HCC,　making　its　assessment　essential　for　diagnosis,　personalized　treatment,　and　prognostic　evaluation.　Multiphoton　microscopy　(MPM),　an　advanced　biomedical　imaging　technology,　capitalizes　on　the　nonlinear　optical　properties　of　biological　tissues,　including　two-photon　excited　fluorescence　(TPEF)　and　second-harmonic　generation　(SHG).　This　allows　MPM　to　achieve　high-resolution,　label-free　imaging　of　tissue　sections,　enabling　precise　visualization　of　tissue　structures　and　co-localized　analysis　of　multiple　tumor-associated　features　within　the　TME.　In　this　study,　we　utilized　MPM　to　perform　label-free　imaging　of　HCC　tissues　and　aimed　to　evaluate　tumor　heterogeneity　within　hepatic　tumor　microenvironment　through　quantitative　image　analysis.　Our　results　demonstrate　that　MPM　images　can　effectively　characterize　tumor　microenvironment,　and　quantitative　calculations　reveal　a　significant　correlation　between　the　tumor　heterogeneity　scores　obtained　by　our　proposed　method　and　tumor　grades.　These　findings　underscore　the　potential　of　MPM　in　investigating　liver　tumor　microenvironment,　suggesting　that　the　heterogeneity　scores　may　aid　in　more　accurate　disease　diagnosis.　©　2025　SPIE.