Detection and visualization of β-galactosidase (β-gal) is essential to reflect its physiological and pathological effects on human health and disease, but it is still challenging to precisely track β-gal in vivo owing to the limitation of current analytical methods. In our work, we reported a photoacoustic (PA) nanoprobe for selective imaging of the endogenous β-gal in vivo. Our nanoprobe Cy7-β-gal-LP was constructed by encapsulation of a near-infra red (NIR) dye Cy7-β-gal within a liposome (LP, DSPE-PEG2000-COOH). The dye Cy7-β-gal was synthesized based on a dye Cy-OH where the hydroxyl group was replaced by a β-D-galactopyranoside residue, which can be recognized by β-gal as an enzyme hydrolytic site. With the addition of β-gal, the absorbance of Cy7-β-gal exhibited a significant red shift with the absorption peak moved from 600 nm to 680 nm, which should generate a switch-on PA signal at 680 nm in the presence of β-gal. In addition, as the fluorescence of the dye was totally quenched due to aggregation within the liposome, Cy7-β-gal-LP exhibited high PA conversion e±ciency. With the nanoprobe, we achieved the selective PA detection and imaging of β-gal in the tumor-bearing mice.