Controlled defects were created on DNA linked 2-D colloidal photonic crystals using a Nd:YAG pulsed laser. The 2-D photonic crystals were self-assembled using 1.8 μm polystyrene (PS) microspheres on functionalized glass substrates. To synthesize the hexagonal close packed crystalline samples, both substrate and particles attached single-strand DNA, sequence A on the substrate and sequence B on the particles. The DNA was hybridized using the DNA linker with complementary single-strand A’B’ that anchored the particles to the substrate during self-assembly. The 532 nm second harmonic wavelength beam of the pulsed Nd:YAG laser (1064 nm) with a pulse width of 10 ns was used for the removal of individual colloidal particles from the self assembled photonic crystals. In the present tests, the diameter of the laser beam was optically reduced from 7 mm to about 1.8 μm. Controlled line defects and geometrical shapes (e.g., hexagonal and triangle) were created in the 2D arrays in an aqueous medium.