Diagnosis of peritoneal metastasis in gastric cancer (GC) is essential for determining appropriate therapeutic strategies and avoiding non-essential laparotomy or gastrectomy. Recently, a variety of activatable fluorescence probes that can detect enzyme activities have been developed for cancer imaging. The aim of this study was to identify the key enzyme involved in peritoneal metastasis in GC. The enzymatic activity of gamma-glutamyl transpeptidase, dipeptidyl peptidase IV, and β-galactosidase (β-Gal) was assessed in lysates prepared from preserved human GC (n = 89) and normal peritoneal (NP; n = 20) samples. β-Gal activity was significantly higher in the human GC samples than in NP samples, whereas no differences were observed in the activities of the other enzymes. Therefore, we used SPiDER-βGal, a fluorescent probe that can be activated by β-Gal, for imaging GC cell lines, peritoneal metastasis in a mouse model, and fresh human resected GC samples (n = 13). All cell lines showed fluorescence after applying SPiDER-βGal, and metastatic nodules in the mice gradually developed high fluorescence that could be visualized with SPiDER-βGal. The human GC samples showed significantly higher fluorescence than NP samples. β-Gal is a useful target enzyme for fluorescence imaging of peritoneal metastasis in GC.