Chiral nanohole array (CNA) films are fabricated by a simple and efficient shadow sphere lithography (SSL) method and achieve label-free enantiodiscrimination of biomolecules and drug molecules at the picogram level. The intrinsic mirror symmetry of the structure is broken by three subsequent depositions onto non-close packed nanosphere monolayers with different polar and azimuthal angles. Giant chiro-optical responses with a transmission as high as 45%, a chirality of 21°μm-1, and a g-factor of 0.17, respectively, are generated, which are among the largest values that have been reported in the literature. Such properties are due to the local rotating current density generated by a surface plasmon polariton as well as a strong local rotating field produced by localized surface plasmon resonance, which leads to the excitation of substantial local superchiral fields. The 70 nm-thick CNAs can achieve label-free enantiodiscrimination of biomolecules and drug molecules at the picogram level as demonstrated experimentally. All these advantages make the CNAs ready for low-cost, high-performance, and ultracompact polarization converters and label-free chiral sensors.