Abstract: Objective To analyze the changes in pathogen constituent of Human enterovirus 71 (EV71) and Coxsachie virus A16 (Cox A16) in children with hand foot and mouth disease(HFMD) in Hangzhou in 2015 and the characteristics of laboratory diagnostic methods of HFMD. Methods A total of 663 children diagnosed with HFMD from April 2015 to August 2015 were enrolled in this study. Enterovirus nucleic acid were detected by fluorescence quantitative RT-PCR and serum antibody of EV71 IgM and Cox A16 IgM were detected by enzyme-linked immunosorbent assay (ELISA). Results Among the 663 children with HFMD, 388 (58.52%) were common nucleic acid positive and 127 (19.16%) were EV71 positive. Compared with the data of 2014, the differences in proportions of common nucleic acid positive group (2=166.306,P=0.000) and the EV71 positive group were significant (2=134.418, P=0.000), and 487 cases (73.45%) were mild ones, mainly in common nucleic acid positive group (65.91%, 321/487), and 176 cases (26.55%) were severe ones, mainly in EV71 positive group(43.75%, 77/176) and common nucleic acid positive group (38.07%, 67/176).The probability of severe cases was different in different groups (2=98.395, P=0.000). The severe case proportion was highest in EV71 group (60.63%, 77/127). The difference in positive rate between nucleic acid detection and serum antibody detection was significant (2=44.487, P=0.000). In 127 cases of EV71 infection, 59.85%(76/127)were positive in both nucleic acid detection and serum antibody detection, 40.15% (51/127) were either nucleic acid positive or serum antibody positive. In 64 cases of Cox A16 infection, 9.38%(6/64) were positive in both nucleic acid detection and serum antibody detection, 90.62%(58/64) were either nucleic acid positive or serum antibody positive. With the quantitative RT-PCR as the standard, the specificity in detection of EV71 and Cox A16 was higher (86.61%), but the sensitivity was lower relatively (69.49%). Conclusion Compared with 2014, the epidemic features of HFMD changed obviously. The detection of virus nucleic acid and antibody can increase the positive rate of laboratory detection, and clinical classification can facilitate the clinical treatment. As the increase of severe cases in common nucleic acid positive group, it is necessary to find a more suitable detection method.