A dual-labeled probe for monitoring caspase activity was designed and synthesized based on a tetraphenylethene (TPE) fluorogen with aggregation-induced emission characteristics and a caspase-specific Asp-Glu-Val-Asp (DEVD) peptide. Two stereoisomers were furnished and successfully separated by HPLC. We demonstrated for the first time the effect of isomerization on the reaction kinetics between the probes and caspase. It was revealed that caspase can produce a much higher light-up ratio for the Z-TPE-2DEVD probe, while its kinetics favor E-TPE-2DEVD due to enhanced probability of optimal binding between the two. Understanding the stereoisomers and their biological functions will open new opportunities for bioprobe design with optimized performance.