The fabrication and supramolecular chirality of complex multilayer films of tetrakis(4-sulfonato-phenyl)porphine (TPPS) and long-chain enantiomers of tryptophan derivatives (D-Trp or L-Trp) were investigated. Two methods were employed to assemble the supramolecular films of Trp with TPPS. In one method, Tip was spread on the aqueous subphase containing TPPS at pH 3.1, and the complex monolayer was formed in situ through the adsorption. The subsequent deposition of the thus-formed monolayer onto solid substrates provided type I complex films. In the other method, a preformed multilayer film of Trp on a solid substrate was immersed into the TPPS solution, and ex situ adsorption afford the formation of the type 11 complex films. A series of techniques such as UV-vis spectroscopy, circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) were used to characterize these complex multilayer films. It has been found that TPPS can form J aggregates and show chirality in both types of multilayer films. It was interesting that the sign of the Cotton effect in type I multilayer films appeared to be undetermined no matter what kind of Tip enantiomer was used. However, the supramolecular chirality of the TPPS J aggregate in type 11 multilayer films was determined by the chirality of Trp. AFM measurement on the films revealed that TPPS formed larger nanorod structures in type I films and produced small granular aggregates in type 11 film. A possible mechanism was proposed on the difference of the chiral monolayer and multilayer film in controlling the chirality of achiral dye molecules.