The pH of lysosome in cells has significant affect to various biological activities, including autophagy, phagocytosis, enzyme processing and so on. The metastasis and apoptosis of cancer cells are closely related to change of the lysosomal pH. The nucleus is the largest organelle in eukaryotic cells which control the most important genetic and metabolic processes, and the enzymes involved in metabolic processes are sensitive to changes of pH. Therefore, it is very important to investigate the pH change in cells. Herein, we designed and synthesized a novel fluorescent probe NpH-1 via a simple two-step reaction. The probe uses naphthalimide as the fluorescent group and morpholine group as the pH responsive site, and regulates the fluorescence through the photoinduced electron transfer (PET) mechanism. The spectroscopic properties of NpH-1 response to pH changes were measured in the Britton-Robison buffers, whose buffering range was from 1.81 to 11.92. In the pH range from 3.0 to 10.0, NpH-1exhibited a very rapid and reversible response to pH change. The pKa of NpH-1 was 5.41. It showed a very high photostability. Moreover, the fluorescence emission of NpH-1 was not influenced by biological species including metal ions, anions and amino acids. NpH-1 showed very low cytotoxicity, which indicated that it could be applied for cell imaging. Thus, we monitored the pH changes in HeLa cells via stimulating them by Chloroquine. The cell imaging results showed that NpH-1 could be utilized for detection of pH changes in living cells. Furthermore, the colocalization experiments of the probe NpH-1 for various organelles including lysosome, mitochondria, Golgi, endoplasmic reticulum and cellular nucleus were carried out, and the results exhibited the probe NpH-1 mainly distributed in lysosome and nucleus, which implied that the probe could be applied for detection of pH change in complex intracellular environment.