The invention discloses eye drops for resisting asthenopia. The eye drops comprise the following components: amiotide eye drops, inosine injection, adenosine disodium triphosphate injection, racanisodamine injection, vitamin B6 injection, taurine eye drops and an assistant for eyes, wherein the assistant for eyes is at least one of injection water or an isoosmotic adjusting agent; the isoosmotic adjusting agent is at least one of a sodium chloride physiological saline, glucose injection, sorbitol eye drops, borate saline buffer, mannitol injection or polyethylene glycol eye drops. The invention also discloses a preparation method of the eye drops for resisting asthenopia. The eye drops have the advantages that: 1, the overall efficacy of a compound preparation is obviously superior to a single drug effect; 2, the eye drops play roles in the whole process of the disease, the dose is reduced, and the side effect is reduced; 3, various medicines work in coordination with each other, are complementary in efficacy, and are taken as preconditions for playing roles; 4, the eye drops are obvious in pharmacological effect, applicable and convenient, and convenient to carry.