To find out the HCN formation process during glycine pyrolysis, the small molecule gaseous pyrolysates, H₂O, NH₃, CO₂, CO, HNCO and HCN, were analysed in real-time with TG-FTIR. The volatile pyrolysis products and solid residues at certain temperature were identified by online Py-two-dimensional GC-MS with heart-cutting and LC-MS/MS, respectively. The pyrolysis of 2,5-diketopiperazine (DKP), which was probably the intermediates of HCN forming from glycine, was studied. The results showed that: 1) The pyrolysis process of glycine could be divided into three temperature ranges: 200-300 °C, 300-440 °C and 440-900 °C, HCN was formed at each range with three peaks appearing at 273 °C, 422 °C and 763 °C; 2) The pathways of HCN formation from glycine at low- and high-temperatures were different. Below 273 °C, glycine underwent a decarboxylation reaction to produce methylamine, which subsequently formed HCN by means of dehydrogenation. Over 300 °C, glycine was dehydrated to form DKP, and then subsequently formed HCN.