In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green Ⅰ was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy (GEC)/μL, the intra- and inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship (r2 = 0.997) between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 100 to 2.31 × 109 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.