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Objective To verify and reveal the mechanism of traditional Chinese medicine (TCM) of Gouqi Juhua Fang (GJ Fang) on asthenopia, this experiment studied the nourishing effect of GJ Fang by establishing the yin deficiency of the liver and kidney pattern model. Methods According to body weight, 50 male Kunming mice were randomly divided into five groups (each n = 10): normal group, model group, Qiju Dihuang Pill (QD Pill) group (3.0 g/kg), low-dose GJ Fang group (65.7 mg/kg), and high-dose GJ Fang group (131.4 mg/kg). Each morning, the intervention groups were administered corresponding medicine by gavage, and the normal group and model group were administered the same amount of distilled water by gavage, for 14 days. Each afternoon, the model group and each intervention group were given levothyroxine sodium solution (150 μg/kg) by gavage, and then the tail was clipped about 3 cm from the tip for 5 minutes, for 14 days. The general state of the mice was observed every day, and all mice were weighed on the 1st, 7th, and 14th day. On the 14th day, the temperature tropism was detected. On the 15th day, all mice were sacrificed, and the samples were collected. The thymus index was calculated; the cyclic nucleotides, thyroid hormones, sex hormones, liver function, and immune factors in serum were detected; the energy metabolism enzyme activities and oxidation levels in the liver tissue were also determined. Results Compared with the normal group, the model group showed symptoms of deficiency and heat such as weight loss, dry and hard stools, yellow urine, increased voluntary activities, irritability, active reactions, and the desire to be close to the cold environment. The symptoms both improved to a certain extent in the QD Pill group and GJ Fang group. Compared with the model group, the low- and high-dose GJ Fang group showed the following: The bodyweight of the mice was increased (P<0.05); the contents of cAMP, cGMP, cAMP/cGMP, E2/T, T4, and ALT were decreased (P < 0.05); the thymus index was increased (P<0.05); and the levels of T3 and IL-2 increased (P<0.05). In addition, the contents of MDA and the activities of Na+-++-ATPase and Ca2+-Mg2+-ATPase decreased (P<0.05) in the liver. Conclusion GJ Fang and QD Pill both can improve internal heat and endocrine hormone disorders in yin deficiency, reduce the rate of energy metabolism, enhance the immunity and antioxidant factors level, and comprehensively relieve the state of yin deficiency in the liver and kidney, thus relieving asthenopia. Because of the safe formula and clear active ingredients, GJ Fang has the potential to be developed into a safe, stable, controllable, and pattern-differentiated product for asthenopia. [ABSTRACT FROM AUTHOR]