Photoacoustic (PA) imaging is advantageous for the diagnosis of superficial cancer with high spatial resolution. However, to the best of our knowledge, using an alkaline phosphatase (ALP)-activatable probe for the enhanced PA imaging of tumors has not been reported. In this work, we rationally designed a NIR probe IR775-Phe-Phe-Tyr(H2PO3)-OH (1P) for PA imaging ALP activity in vitro and in tumor. Under the catalysis of ALP, 1P was efficiently converted to IR775-Phe-Phe-Tyr-OH (1), which self-assembled into the nanoparticles 1-NPs. The formation of 1-NPs induced a 6.4-fold enhancement of the 795 nm PA signal of 1P. In vivo tumor PA imaging results indicated that, compared to that in the ALP inhibitor-treated control group, PA contrast in the experimental group enhanced 2.3 folds at 4 h after 1P injection. By replacing the Phe-Phe-Tyr(H2PO3)-OH motif in 1P with other enzyme-cleavable ones, we hope that more PA probes could be developed for the precise diagnoses of their corresponding cancers in the near future.