Polymethyl methacrylate (PMMA) has emerged as a popular substrate due to its affordability, ease of use, and compatibility with human tissue. Laser micro-machining, particularly with CO2 lasers, is favored for its speed, flexibility, and cost-effectiveness in creating microchannels. It’s crucial to understand how machining settings affect performance, especially for heat-sensitive materials like PMMA. This paper explores the impact of laser power, scanning speed, and their ratio on the depth, width, and heat-affected zone (HAZ) in PMMA microchannels. Eleven experimental runs designed by Design Expert software using central composite design, where power and scanning speed varied from 20 to 80 W and 200 to 500 mm/s, respectively, were conducted using a CO2 CNC laser machine. The channel depth, width, and HAZ were measured using an optical microscope, and finite element analysis was used to couple temperature-dependent material properties and laser parameters to predict channel dimensions and HAZ. The response model was developed and optimized using the response surface method. The minimum HAZ zone, 0.104 mm, was achieved at 47 W and 500 mm/s scanning speed while getting 0.097 mm depth and 0.3 mm width. Finite elemental results and optimization results closely match the experimental run. Hence, this prediction procedure can be used to design microchannels with minimal HAZ.