A caspase-7 biosensor (vDEVDc) based on FRET (fluorescence resonance energy transfer) was used to study the proteolytic properties of caspase-7, an executioner protease in cellular apoptosis. An active isoform of caspase-7 with the 56 N-terminal residues truncated (57casp7) cleaved vDEVDc at the recognition sequence, resulting in a FRET efficiency decrease of 61%. In contrast, an isoform with the 23 N-terminal residues truncated (24casp7) bound to vDEVDc but did not cleave the substrate, resulting in a FRET increase of 15%. Kinetic results showed an exponential substrate cleavage and binding curve for the 57casp7 and 24casp7 isoforms, respectively. FRET changes of the vDEVDc biosensor were also monitored in cos-7 cells upon STS-induced apoptosis. Finally, we modeled caspase-7 binding to vDEVDc and estimated a FRET emission ratio increase of 31.7%, which agrees with the 15% experimental result. We showed that two differently truncated isoforms of caspase-7 exhibit different enzymatic properties, namely binding by 24casp7 and hydrolysis by 57casp7.