Caspase-3 is an essential executor in apoptosis, and its activation has been regarded as a biomarker of cell apoptosis. The development of Caspase-3-responsive multimodal probes is a promising research prospect. Fluorescent/photoacoustic (FL/PA) imaging has attracted considerable attention due to the high sensitivity of FL as well as the high spatial resolution and penetration depth of PA. To our knowledge, there has been no tumor-targeted FL/PA probe to monitor the activity of Caspase-3 in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Bio-DEVD-HCy) for Caspase-3-responsive imaging of tumor apoptosis. Ac-DEVD-HCy without tumor-targeted biotin is used as a control probe. In vitro experiments indicated that Bio-DEVD-HCy is superior to Ac-DEVD-HCy because of the higher kinetic parameter of Bio-DEVD-HCy in comparison to Ac-DEVD-HCy. Cell and tumor imaging results suggested that Bio-DEVD-HCy could enter and accumulate in tumor cells with higher FL/PA signal with the help of tumor-targeted biotin. In detail, Bio-DEVD-HCy or Ac-DEVD-HCy could image apoptotic tumor cells with 4.3-fold or 3.5-fold FL enhancement and 3.4-fold or 1.5-fold PA enhancement. Bio-DEVD-HCy or Ac-DEVD-HCy could image tumor apoptosis with 2.5-fold or 1.6-fold FL enhancement and 4.1-fold or 1.9-fold PA enhancement. We envision that Bio-DEVD-HCy will be applied for FL/PA imaging of tumor apoptosis in clinical settings.