Viral nanoparticles have attracted extensive research interests in diverse applications of diagnosis and therapy. In particular, filamentous M13 bacteriophages have shown great potential in biomedical applications. However, its pathways entering into cells still remain unclear, and this greatly hinders its further use as a drug or gene carrier. Here, a ratiometric M13 pH probe is designed by conjugating two fluorescent dyes onto the surface of M13. Since the intensity ratio is not influenced by probe concentration, ion strength, temperature, photobleaching, and optical path length, this ratiometric probe can be used to investigate the intracellular pH map of M13. More importantly, the internalization mechanism of M13 can be elucidated. It is found that this filamentous phage shows great cell-type dependence in interaction with cells and internalization mechanism. The phage tends to be bounded on the cell membrane of only epithelial cells, not endothelial cells. Furthermore, the M13 phage enters into cells through endocytosis with specific mechanism: clathrin-mediated endocytosis and macropinocytosis for HeLa; vesicular transport, clathrin-mediated endocytosis, and macropinocytosis for MCF-7; caveolae-mediated endocytosis for human dermal microvascular endothelial cell (HDMEC). This work provides key notes for cancer diagnosis and therapy based on filamentous bacteriophage, especially for design of pH-sensitive drug delivery systems.