The aggregation and induced chirality of an anionic meso-tetraphenylsulfonato porphyrin (TPPS) on a layer-by-layer assembled deoxyribonucleic acid/poly(allylamine hydrochloride) (DNA/PAH) film were investigated. It was found that TPPS could directly penetrate into a DNA/PAH film and be assembled into both H-aggregates and J-aggregates. Both of the aggregates showed strong induced circular dichroism (CD) in the film. On the other hand, TPPS could also penetrate into the DNA/PAH film through the help of a cationic meso-tetrakis(4-N-methylpyridyl) porphyrin (TMPyP), as a spacer. In this case, J-aggregates formed in the DNA/PAH matrix and induced chirality was also observed. The induced chirality of the TPPS J-aggregate followed the DNA chirality, in the presence of TMPyP; however, an opposite induced chirality was observed in the absence of TMPyP. Atomic force microscopy images showed nanorods on the surface of DNA layer in both cases, which were composed of H-type and/or J-type aggregates of TPPS. The mechanism of the formation of the opposite induced CD signals was discussed.