The fabrication of functional assemblies with defined structures through controllable molecular packing under physiological conditions is challenging. Here, modularly designed peptide‐cyanine conjugates that intracellularly self‐assembly into 1D columnar superstructures with controlled cyanine aggregation were designed, and they exhibit distinct imaging or photothermal properties. The peptide backbone is cleaved by caspase‐3/7 after entering the cells. Then the self‐assembled residue, with a double cyanine substitution (Pr‐2Cy), forms a P helical column in which H‐aggregated cyanine dyes show 3.4‐fold photothermal conversion efficiency compared to free ones. The self‐assembled residue with a single cyanine substitution (Pr‐1Cy) forms a loose column, in which cyanine dyes with undefined structure have a fluorescence quantum yield of up to 9.5 % (emission at 819 nm in H2O). This work provides a simple way to modify in vivo self‐assembled peptides with functional molecules for achieving desired bio‐applications. The design of peptide‐cyanine conjugates that intracellularly self‐assemble into a one‐dimensional column with controlled cyanine aggregation exhibit distinct imaging or photothermal properties. Through rational molecular design, the assembly behavior of cyanine dyes is precisely regulated in biological systems, resulting in two kinds of assemblies with different properties. The relationship between structure and function has been studied in detail.