Macrophages　are　at　the　center　of　the　invasion　and　play　a　protumoral　role　in　the　tumor　microenvironment.　They　can　facilitate　angiogenesis,　extracellular　matrix　remodeling,　invasion　and　migration　of　cancer　cells,　while　inhibit　the　antitumoral　immune　surveillance.　Tumor-associated　macrophages　are　a　major　component　of　malignant　tumors,　which　can　stimulate　the　progress　of　tumors.　In　many　human　cancers,　the　presence　of　macrophages　in　tumor　microenvironment　has　been　correlated　with　unfavorable　prognosis.　Therefore,　visual　diagnosis　of　macrophages　in　tumor　microenvironment　is　great　significance　for　us　to　comprehend　the　occurrence　and　progress　of　tumor.　Multiphoton　microscopy　(MPM)　with　subcellular　resolution　based　on　second　harmonic　generation　(SHG)　and　two-photon　excited　fluorescence　(TPEF)　is　very　suitable　for　real-time　detecting　morphological　and　structural　changes　in　biological　tissues　without　tissue　staining　and　exogenous　probe　molecule.　In　this　study,　we　describe　the　use　of　label-free　MPM　for　analyzing　the　intratumoral　distribution　and　morphological　changes　of　macrophages　in　breast　tumor　microenvironment.　Our　results　indicated　that　MPM　can　accurately　identify　macrophages　in　breast　tumor　tissues.　MPM　images　were　well　consistent　hematoxylin　and　eosin　(H&E)　stained　images.　This　work　would　provide　the　basis　for　further　quantifying　these　changes　using　MPM.　With　the　development　of　miniaturized　imaging　devices,　MPM　may　be　a　promising　imaging　technique　for　clinicians　to　study　various　structural　features　in　tumor　microenvironment.　©　2019　SPIE.