Lymphovascular invasion (LVI) and perineural invasion (PNI) are 2 important pathologic parameters and need to be accurately assessed in multiple malignancies. Integrin β4, a member of the integrin family, has been reported to be positively expressed in vascular endothelia, peripheral nerves, and a collection of epithelia. However, little is known about the effectiveness of β4 immunostaining on the recognition of LVI and PNI. Herein, we explored the applicability of β4 immunostaining in stomach, thyroid, and breast cancers. Parallel immunostaining of D2-40, CD34, and S-100 was performed as controls for lymphatic endothelia, vascular endothelia, and neural fibers, respectively. The results demonstrated that β4 concurrently stained the lymphatic and vascular endothelia, and the peripheral nerves. Both LVI and PNI were clearly and accurately outlined by β4 immunostaining. β4 was also expressed in the majority of tumor cells, enabling recognition of LVI and PNI encroached by small tumor clusters. In contrast to D2-40 and CD34, β4 staining was not observed in stromal cells, and therefore it facilitated differentiation between the shrinkage cleft and LVI. According to our results, β4 staining strikingly increased the diagnostic accuracy and interobserver concordance for LVI and PNI compared with hematoxylin and eosin staining alone. Finally, the applicability of β4 was confirmed in 9 other types of malignancies, including cancers of the colon, prostate, esophagus, lung, kidney, uterus, tongue, bladder, and liver. Collectively, β4 is a reliable marker for synchronous detection and diagnosis of LVI and PNI.