The embodiment of the invention discloses a nucleic acid fluorescence PCR detection kit for a universal enterovirus, a coxsackievirus A16 and an enterovirus 71. The nucleic acid fluorescence PCR detection kit comprises an RNA extracting solution, an RNA eluant, internal standards, a PCR reaction solution, an EV/CA16/EV71 enzyme mixed solution, EV/CA16/EV71 positive reference substances and EV/CA16/EV71 negative reference substances. According to the kit, the universal enterovirus, the coxsackievirus A16 and the enterovirus 71 can be simultaneously detected in a same sample, but other pathogen RNA cannot be detected, the detection sensitivity can reach 400 copie/ml, the detection range is 4.0 E+02-4.0E+08 copies/ml, and a reliable experimental evidence is supplied to early diagnosis on infection of the universal enterovirus, the coxsackievirus A16 and the enterovirus 71.