Photobiomodulation is a practical and non-invasive treatment that triggers cell proliferation, cell differentiation, wound healing, new tissue formation, inflammation and pain reduction with low-level light therapy. Light-emitting diodes (LEDs) are energy-saving, affordable and safe alternatives to laser devices which are recently preferred in photobiomodulation. Although the wavelengths between 600–700 nm are most preferred ones, there is a lack of practical optical systems which study this mechanism in vitro with different wavelengths simultaneously. In this study, a portable and remotely controlled multicolor LED-based system was designed and tested on the wound healing process of human keratinocytes by irradiating the cells homogenously with 3 different wavelengths (460–475 nm as blue, 515–535 nm as green, and 585–595 nm as orange) on different experimental groups at the same time. Its proliferative and wound healing effect was evaluated with cell viability (MTT) analysis and cell migration (scratch) assay, respectively. It was observed that orange-LEDs were designated as the most triggering wavelength in terms of cell proliferation. Also, it was revealed with this device that different wavelengths can reach the intended accelerated wound healing process, so this optical system will be an advantageous design for future practical photobiomodulation studies in vitro.