Fluorescence imaging of tumours facilitates rapid intraoperative diagnosis. Thus far, a promising activatable fluorescence probe for hepatocellular carcinoma (HCC) has not been developed. Herein, the utility of the fluorescence imaging of HCC using a β-galactosidase (β-Gal)-activatable fluorescence probe SPiDER-βGal was examined. β-Gal activity was measured in cryopreserved tissues from 68 patients. Live cell imaging of HCC cell lines and imaging of tumour-bearing model mice were performed using SPiDER-βGal. Furthermore, fluorescence imaging was performed in 27 freshly resected human HCC specimens. In cryopreserved samples, β-Gal activity was significantly higher in tumour tissues than in non-tumour tissues. Fluorescence was observed in HCC cell lines. In mouse models, tumours displayed stronger fluorescence than normal liver tissue. In freshly resected specimens, fluorescence intensity in the tumour was significantly higher than that in non-tumour liver specimens as early as 2 min after spraying. Receiver operating characteristic curves were generated to determine the diagnostic value of SPiDER-βGal 10 min after its spraying; an area under the curve of 0.864, sensitivity of 85.2%, and specificity of 74.1% were observed for SPiDER-βGal. SPiDER-βGal is useful for the rapid fluorescence imaging of HCC. Fluorescence imaging guided by SPiDER-βGal would help surgeons detect tumours rapidly and achieve complete liver resection.