In this paper, we investigated the optical output characteristic of the light guide plate (LGP) with microstructures engraved by a CO2 laser, which is for edge-lit backlight of liquid crystal display or illumination. Especially, the laser-engraving method is suitable for the slim large-sized LGP used in LED TV backlight, and the engraved microstructure has polished surface to contribute to better optical efficiency. For seeking higher optical efficiency, we adopted a LGP with lenticular-arrayed surface (LAS LGP) as the experimental substrate. In order to investigate the effect of the lenticular-arrayed surface on optical output characteristic for the different kinds of the engraved microstructures, we used the laser to directly engrave the flat surface (opposite to the lenticular-arrayed surface) of the LAS LGP with different kinds of microstructures as experimental samples; each sample has one kind of the engraved microstructures on its flat surface. Similarly, we also engraved the same kinds of the microstructures on the flat LGP as a controlled group for comparison. The cross-section profiles of the engraved microstructures on both LAS LGP and flat LGP were measured by a laser confocal microscope. All the samples were further measured for spatial and angular luminance by the BM7 and Conoscope, respectively. In addition, both the experimental data and simulation results were demonstrated and compared to each other in this paper.