Objective: To explore the differences among three methods of nucleic acid extraction and three kinds of real-time fluorescence quantitative PCR instrument. Methods: Twenty-five respiratory virus nucleic acid and 25 enterovirus nucleic acid positive samples were with selected at random and nucleic acids were extracted by using three methods (method A, B, and C). The results among different methods were analyzed by randomized block design. 25 respiratory viral nucleic acid positive specimens and enterovirus nucleic acid positive samples were detected by using three kinds of real-time fluorescence quantitative PCR instrument (instrument A, B, and C). The results among different instruments were analyzed by randomized block design. Results: There was a significant difference among three methods of nucleic acid extraction in results(χ²=42.9162, P<0.001), in which method A and C had not significant difference(Z=0.837, P=0.3816>0.05), while method A vs. B, B vs. C were significantly different(Z=7.025, P<0.001; Z=7.9, P<0.001). There was also a significant difference among three kinds of real-time fluorescence quantitative PCR instrument in results(χ²=23.773, P<0.001), in which instrument B and C had no significant difference(Z=0.75, P=0.4533>0.05), while instrument A vs. B, A vs. C were significantly different(Z=5.70, P<0.001; Z=6.45, P<0.001). Conclusions There is difference among different methods and instruments in the test results under the same condition, which call for options in practical work according to need.