Polypyrrole (PPy) was synthesized by a galvanostatic method using (1 S)-(+)-10-camphorsulfonic acid ((+)-CSA) as the dopant, and the produced PPy was further overoxidized in a solution of (+)-CSA. A chiral microenvironment was successfully formed in the overoxidized PPy (OPPy) as a result of the synergistic effects of overoxidation and (+)-CSA, resulting in a twisted helical architecture of the OPPy chains. The formation of optically active OPPy was confirmed from aspects of its morphology (SEM and AFM) and circular-dichroism (CD) spectra. Finally, an electrochemical chirality sensor was fabricated on the basis of the resultant OPPy, which exhibited excellent biomolecular homochirality in the discrimination of tryptophan (Trp) enantiomers.