Apoptosis is a tightly regulated biochemical process essential for cellular homeostasis, in which the mitochondrial release of Cytochrome c (Cyt c) represents an early and irreversible hallmark. Reliable detection of Cyt c is therefore of great importance in apoptosis-based drug screening and cancer research. In this study, we report a label-free colorimetric aptasensor for Cyt c detection based on the in-situ synthesis of gold nanoparticles (AuNPs) on a polyguanine (poly-G)-extended aptamer, without any chemical modification or DNA labeling. The aptamer-templated AuNPs (Apt@AuNPs) act as both a recognition element and signal transducer, where specific binding of Cyt c induces nanoparticle aggregation, leading to a distinct localized surface plasmon resonance (LSPR) red shift that is visible to the naked eye. The proposed sensing platform exhibits a linear response toward Cyt c in the concentration range of 1 to 1000 µM, with a calculated detection limit of 0.47 µM. In addition the intrinsic nanozyme activity of Apt@AuNPs was exploited to develop a dot blot assay for selective Cyt c recognition. Importantly, the assay successfully differentiated apoptotic and non-apoptotic cancer cells based on a clear color change (purple vs. red). Overall, this work introduces a simple, modification-free, and visually interpretable biosensing strategy for Cyt c detection, highlighting its potential application in apoptosis assessment and apoptosis-based drug screening, particularly in settings requiring rapid and instrument-free analysis.