Two-photon fluorescence scanning confocal microscopy sensitive to circular dichroism with a diffraction-limited resolution well below 500 nm is demonstrated. With this method, the spatial variation of the circular dichroism of thermally annealed chiral polyfluorene thin films has been imaged. We observed circular dichroism associated with submicrometer-sized domains showing helicoidally twisted macromolecular organization. Domains with opposite chiroptical properties, corresponding to left- or right-handed molecular organization, coexist in the film. Our results are consistent with those obtained by one-photon imaging and illustrate the potential of two-photon imaging for use in studying helical macromolecular organization.