The field of microfluidics has rapidly evolved and has become pivotal in enabling precise control and manipulation of fluids at the microscale, with applications across various domains such as chemical synthesis and biomedical diagnostics. However, equipment to accurately observe and measure microfluidic channel samples is not available, especially in the research conditions of developing countries. This study proposed a novel technique to quantitatively evaluate the depth of microfluidic channels using optical microscopy. Taking advantage of optical microscopy, including ease of use, dissemination, and cost-effectiveness, the method involves acquiring microscopy images followed by rigorous image analysis techniques to extract pertinent geometrical data. Specimens are prepared by Polymethyl Methacrylate (PMMA) sheets to simulate preformed microfluidic channels of known depths. Subsequently, optical microscopy is employed to measure desired dimensions, such as the depth of the channels at numerous points, to assess their conditions and shapes. These measurements are then compared with the predetermined dimensions to evaluate the method’s accuracy. A dataset of depth measurements obtained multiple times on the microfluidic channels is compiled, allowing the calculation of the average depth along with the associated error and standard deviation of the optical microscopy method. Furthermore, the measurement is repeated multiple times along the length of the microfluidic channel, thus evaluating the shape of the channel. Comparisons are made between the obtained results and the initially provided dimensions to investigate the method’s accuracy, thereby concluding the potential of the proposed optical microscopy-based depth measurement method and new directions for its development in the biomedical field.