Herein, the cobalt hydroxide is firstly prepared by hydrothermal method using cobalt chloride (CoCl2·6H2O), polyethyleneimine (BPEI), and octylamine. Then, the Co3O4 is prepared by thermal decomposition of cobalt hydroxide. The Co3O4/glass carbon electrode (Co3O4/GCE) is fabricated by dropping Co3O4 suspension on surface of GCE and is applied to detect the hydrogen peroxide (H2O2) through the cyclic voltammetry and timing ampere response. The effects of calcination temperature on shape of Co3O4 and performance of Co3O4/GCE to detect H2O2 are systematically studied. The results demonstrate that the Co3O4 prepared at annealing temperature of 200℃ presents a hexagonal nanoplates shape and the 200-Co3O4/GCE shows the highest response current value to H2O2. The current response of the 200-Co3O4/GCE has a good linear relationship with the concentration of H2O2 in the range of 0.01~0.20 mmol/L, the detection limit is 6.53 μmol/L (S/N=3), and the sensitivity is 875.66 μA/(mmol·L-1·cm2). In addition, 200-Co3O4/GCE also shows a good anti-interference ability for H2O2 detection.