Potato is an important crop in terms of its nutritional value, cost-effective production, versatile utilization, and starch production. To produce disease-free potato starting material, the production of microtubers in vitro is a viable solution. The process of tuberization is a multifaceted physiological phenomenon that is regulated by a multitude of factors, including environmental conditions, nitrogen availability, plant growth regulators (PGRs), genetic makeup, nutrient availability, photoperiodic, temperature, explant source, potato cultivar, and sucrose. In this study, we evaluated the impact of diverse light qualities and photoperiods on in vitro microtuberzation of potato single node explant. Based on our finding, in comparison with white (W: control), the inclusion of the far-red wavelength in a red-blue light (RBF) in prolonged photoperiod significantly increased both tuberization percentage (18%) and tuberization degree (60%) in Sante (R) cultivar. Additionally, in comparison with W light at 16 h photoperiod, the application of RBF led to significant increment in various parameters, such as explant fresh weight (23%), microtuber diameter (40%), microtuber number (47%), and microtuber fresh weight (121%), microtuber dry weight (166%), and microtuber yield (FW and DW; 227%). Similarly, the utilization of the far-red spectrum in combination with the red-blue spectrum resulted in a 27% increase in protein content compared to W (control).