We report nearly diffraction-limited lasing from the two-dimensional distributed Bragg reflector (DBR) resonator with a conjugated polymer layer as the gain medium. The resonator consists of four Bragg mirrors, which are fabricated by dot-matrix holography. The in-plane optical confinement in this cavity is achieved via two-dimensional Bragg reflections and the vertical confinement is provided through total internal reflection at the slab interface, leading to a three-dimensional confinement of the emitter. Upon optical pumping, the cavity emits nearly diffraction limited laser emission perpendicular to the surface with a threshold of 9 μJ/cm2 and a bandwidth of 0.31 nm which is quite advantageous to its one-dimensional counterpart. Facile fabrication of two-dimensional DBR polymer lasers with different parameters by dot-matrix holography was also demonstrated. It was further found that the cavity Q factor decreases by a factor of two and the laser threshold increases to 0.41 mJ/cm2 when the mirror size decreases to 20 μm. Our results indicate a step towards the application of polymer micro-lasers in the fields of displays and spectroscopic analyzing.