The invention discloses a hand-foot-mouth disease virus EV71 nucleic acid detection kit, and belongs to the technical field of virus detection. The kit consists of N.BstNBI enzyme, T7 RNA (Ribonucleic Acid) polymerase, AMV (Avian Myeloblastosis Virus) reverse transcriptase, RNase H, NASBA (Nucleic Acid Sequence Based Amplification) reaction solution, a NASBA reaction primer, an RIDA (Radioisotope Dilution Assay) probe, DEPC (Diethypyrocarbonate) water, influenza virus a HINI RNA, breast cancer cell MCF-7 total RNA and EV71 RNA. According to the kit, a hand-foot-mouth disease virus EV71 nucleic acid sequence is detected by combining NASBA and RIDA technologies; and the kit has the advantages of sensitivity, specificity, easiness, convenience, high speed, high efficiency, no dependency on special instruments and the like.