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Light-emitting diodes (LEDs) are currently being used as a light source for in vitro regeneration or the growth of plants in a controlled environment. However, it is crucial to define the target system’s sensitivity to light quality before LEDs may be employed as the exclusive source of light. The objective of the present work was to investigate the influence of red (R), blue (B), white (W), and a combination of red plus blue (1:1) and red, blue, and green (1:1:1) LEDs on Gerbera jamesonii cv. ‘Shy Pink’ during in vitro regeneration. It was observed that LED lighting had a substantial impact on the process of shoot regeneration, shoot growth, and rooting of the shoots. When compared to other light treatments, red plus blue (1:1) LED had the greatest impact on the regeneration of shoots, growth of shoots, and root regeneration from shoot and root growth. Length of shoots, height of plantlets, and petiole length were affected by a red LED, and leaf length, width, and area were affected by a blue LED. The content of photosynthetic pigments (Chl a, Chl b, total chlorophyll, and carotenoids) was higher with acclimatized plants upon transplantation, which were regenerated under blue LEDs. In acclimatized plants, photosynthetic efficiency was calculated. Higher internal CO2 concentrations (Ci), photosynthetic rates (Pn), stomatal conductance (Gs), and transpiration rates (Tr) were seen in plants that were grown under red plus blue (1:1) LED. However, the plants that were grown under white LEDs had higher chlorophyll fluorescence (Fv/Fm). Collectively, the current results suggest that red and blue LED is suitable for in vitro regeneration of Gerbera jamesonii cv. ‘Shy Pink’ plants compared to red, blue, and white LEDs.