Four substances (theanine, gamma-aminobutyric acid, beta-carotene and grape seed extract) were added into the chocolate and the improvement effect of these substances on asthenopia of 102 test persons were studied by questionnaire, vision test, attention test, memory test and brain wave test for 30 days. The questionnaire results showed that the total and single score (ocular discomfort score and general malaise score) of sample group were lower than the score before eating the sample chocolate, which is indicated that the asthenopia was improved by eating the sample chocolate, for example, the score of blurred vision was decreased by 0.21 and the score of subjective memory loss was decreased by 0.46 after eating the sample chocolate for 7 days. Attention test and memory test results showed that the attention and memory of sample group were improved significantly with cumulative effects and short-term effects, respectively. And the Sult square time and brain age were decreased by 5.09 s and 2.49, respectively. In the brain wave test, the alpha wave power value, the percentage of the alpha wave power value spectrum and the percentage ratio of the α/β wave power value spectrum which reflect the relaxation degree of the brain in the Lead P4 and Lead O2 of the sample group were increased, and the Lead P4 increased 7.69, 0.04 and 0.50, the Lead O2 increased by 9.90, 0.04 and 0.73, respectively. Therefore, a chocolate product containing theanine, gamma-aminobutyric acid, beta-carotene and grape seed extract plays a mitigation role on asthenopia.