We have introduced line defects into a three-dimensional (3D) photonic crystal by combining the techniques of optical photolithography, self-assembly, and template replication. The template is a colloidal crystal film that is grown on a silicon or glass substrate patterned with photoresist lines, and the 3D photonic crystal with air-core line defects is obtained by infiltrating the colloidal crystal film with silica and removing the template by calcination. SEM and optical transmission spectra show that the resulting 3D PC structure has high structural integrity and crystalline quality. This approach can introduce defects at the desired places of 3D photonic crystals, which may be useful in making 3D photonic crystal-based air-core waveguides.