Objective: To investigate the etiology composition of enterovirus (EV) in patients with severe hand, foot, and mouth disease (HFMD) in children. To assess the diagnostic value of cerebrospinal fluid (CSF) tests in severe HFMD, and to find the key laboratory tests for severe HFMD. Methods: A total of 288 hospitalized cases of children clinically diagnosed with severe HFMD in Hangzhou Children′s Hospital were included from March to July 2016. Throat swabs were collected and enterovirus nucleic acids were detected by fluorescence quantitative reverse transcription (RT)-PCR. Synchronous CSF and serum samples were collected for EV-A71 and CV-sackievirus A16 (CV-A16)-IgM antibody detection. CSF samples underwent routine and biochemical tests. Normally distributed continuous variables were compared using t test. Non-normal distribution continuous variables were compared using Mann-Whitney U test. Differences between categorical variables were compared with χ² test. Results: The total positive rate of enterovirus nucleic acid EV-A71/CV-A16/EV by fluorescence quantitative RT-PCR in the 288 cases of children clinically diagnosed with HFMD was 83.7% (241/288), including EV-A71 55.2% (159/288), CV-A16 4.9% (14/288) and the other enterovirus 23.6% (68/288). Among the other enterovirus group, there were 29.4% CV-A6 (20/68) , 16.2% CV-A4 (11/68) and CV-A10 10.3% (7/68). The total positive rate of combined serum and CSF detection of EV nucleic acid and EV-A71 and CV-A16 IgM antibody was 98.3% (283/288). EV nucleic acid positive rate was 83.7% (24/288). The positive rates were statistically different (χ² =37.289, P=0.000). The CSF nucleated cells count in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (Z=-4.472 and -9.991, respectively, both P<0.05). The CSF nucleated cells positive rate in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (χ²=43.857 and 133.078, respectively, both P<0.05). The CSF protein level in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (Z=-3.151 and -5.255, respectively, both P<0.05). The CSF protein positive (>400 mg/L) rate in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (χ²=4.956 and 11.795, respectively, both P<0.05). The CSF nucleated cell counts and positive rates in EV-A71 IgM antibody-positive subgroup were both higher than those in antibody-negative subgroup (both P<0.05). The CSF protein level and elevated proportion in antibody-positive subgroup were both higher than those in antibody-negative subgroup (both P<0.05). The lactate dehydrogenase concentration in antibody-positive subgroup was significantly higher than those in antibody-negative subgroup (P<0.05). The EV-A71 IgM antibody in serum was significantly correlated with the antibody in CSF (r=0.600, P<0.05). Conclusions EV-A71 is still the most important pathogen of severe HFMD in Hangzhou in 2016. Other enterovirus such as CV-A6, CV-A4 increases compared to those in 2014 and 2015. The CSF routine and biochemical tests and the IgM antibody levels can serve as an important indicator for the diagnosis of children with severe HFMD.