Lysosomal pH is closely related to the metastasis and apoptosis of cancer cells. Detecting lysosomal pH changes in cancer cells could be helpful for analyzing tumor progressions and in-depth study of the roles of lysosomes in tumor invasion and metastasis. Herein, we describe a novel tumor-targeting and lysosome-specific two-photon fluorescent probe (BN-lys) for imaging pH changes for the first time. Biotin was employed as the tumor-targeting module, and morpholine was selected as the lysosome-specific group and the pH site to control the fluorescence by photoinduced electron transfer (PET) mechanism. With a pK_a value of 5.36, BN-lys showed a fast and reversible fluorescence response to pH. Under the guidance of the biotin group, BN-lys displayed strong one-photon and two-photon fluorescence responses to lysosomal pH in cancer cells, while it displayed weak fluorescence in normal cells. Furthermore, BN-lys could be applied for the imaging of chloroquine-stimulated lysosomal pH changes in living cells. These features demonstrate that this probe could have practical applications in biological research.