Dendrobium hybrids have a significant role in the present floral sector. The aim of this research was to evaluate how various light qualities affect the physiological and biochemical traits of Dendrobium ‘Shuijing’. In order to determine the optimal light quality for in vitro cultivation of Dendrobium plantlets, we examined the correlations between growth, antioxidant capacity, and nutrient and chlorophyll levels, as well as chlorophyll fluorescence. The growth rate was compared by using different light qualities emitted by the LED light source. These included red light (R), blue light (B), and three ratios: 8R:2B, 7R:3B, and equal proportions of both colors, known as white, fluorescent light (CK). The combination of 7R:3B resulted in noticeable enhancements in leaf count, root length, root activity, fresh and dry weight measurements, antioxidant capability, as well as chlorophyll content and fluorescence. Specifically, the mixture of red and blue LED lights at a ratio of 7R:3B led to increased leaf number, root length, root activity, fresh and dry weight measurements, antioxidant ability, and chlorophyll content with improved fluorescence. In order to explore the effect of light quality on the growth and development of Dendrobium, the chlorophyll content and chlorophyll fluorescence parameters of plants under all light quality conditions were analyzed by using a linear regression model with other physiological and biochemical indexes. A significant correlation between non-photochemical quenching (NPQ) and leaf length was also observed. The content of chlorophyll b showed significant correlations with both root number and leaf number. Furthermore, chlorophyll a, along with its ratio to chlorophyll b, significantly correlated with root length. Chlorophyll b and the relative electron transport rate of PSII (ETRII) significantly correlated with root activity and the free proline content (FPC) and catalase (CAT) activity. The photochemical quenching coefficient (qP) significantly correlated with total soluble sugars content (SSC) and peroxidase (POD) activity. The correlation between the quantum yield of PSII (Fv/Fm ratio) and superoxide dismutase (SOD) activity was found to be significant. Similarly, the effective quantum yield of PSII photochemistry (ΦPSII) showed significant correlations with fresh weight, dry weight, soluble protein content (SPC), and ascorbate peroxidase (APX) activity. Through a principal component analysis (PCA), it was observed that plants cultivated under the 7R:3B light treatment achieved significantly better comprehensive scores compared to those grown under different light treatments. In conclusion, growth achieved under an LED emitting a ratio of 7R:3B light yielded the most robust Dendrobium hybrid plantlets within a controlled environment.