The invention discloses a nucleic acid test standard substance for rotavirus group A and a preparation method and application thereof. The nucleic acid test standard substance for rotavirus group A has an RNA sequence shown in SEQ ID No. 1. The invention also particularly discloses a preparation method of the nucleic acid test standard substance for rotavirus group A and application of the nucleicacid test standard substance for rotavirus group A in rotavirus group A nucleic acid test. The nucleic acid test standard substance for rotavirus group A is free of biological contamination, easy toprepare and good in uniformity and stability, and has accurate and traceable traits; the nucleic acid test standard substance for rotavirus group A can serve as a positive control for qualitative nucleic acid test of rotavirus group A, may also serve as an external standard for quantitative nucleic acid test of rotavirus group A and may also serve for evaluating novel nucleic acid test methods ofrotavirus group A, material basis can be provided for the certification and accreditation of nucleic acid test capacity for rotavirus group A, and the nucleic acid test standard substance for rotavirus group A is widely applicable to various labs.