The traditional laser drilling method is inefficient and slow. Preparation of medical titanium alloy microhole substrate by laser high-speed polygon mirror can prepare tens of thousands of microhole structures within 100 s. We primarily focused on the preparation of medical titanium alloy microhole substrates using a high-speed polygon mirror. The effects of laser parameters and the polygon mirror’s scanning speed on the drilling aperture, depth, taper, and hole morphology were investigated. The high-repetition-rate laser with a power of 500 W yields a hole diameter of 100 μm, depth of 156 μm, and zero taper. The maximum scanning speed of the high-speed polygon mirror reaches 1000 m/s. Before processing, hole spacing, row spacing, and other related parameters were determined. A processing efficiency of 10000 holes per second is achieved. In addition, to increase the machining aperture, an optimized machining technology was proposed to correct the scanning position deviation of the polygon mirror during the scanning process. A final hole diameter of 150 μm can be achieved using this method. In addition, this method is beneficial to biomaterial coatings and deposit drugs on the titanium alloy microhole substrates.