A novel LED-based light source is developed for the photodynamic therapy (PDT) treatment of cells in vitro. According to the characteristics of porphyrin-based photosensitizers for PDT applications, the red LED with emission wavelength of 627 nm is chosen. The arrangement mode and the distance between two LED units are optimized based on the simulation of light distribution for LED array by using TracePro software. Furthermore, the secondary optical design is performed in order to further improve the illumination uniformity. Based on the mathematical simulation, a novel LED-based light source is successfully developed, and the average power density and illumination uniformity are measured to be around 13.7 mW/cm2 and 90%, respectively. The obtained results imply that the LED-based light source is a convenient and promising light source for in vitro PDT studies.