(2E)-Hexenal is widely used in flavors and perfumes; however, it is mainly derived from chemical synthesis. In this study, hydroperoxide lyase (HPL) naturally originated from Amaranthus tricolor was used to catalyze the reaction to generate (2E)-hexenal from 13-hydroperoxy-9Z, 11E, 15 Z-octadecatrienoic acid (13-HPOT), and salt-adding steam distillation was used for the separation of (2E)-hexenal. A maximum yield of (2E)-hexenal that reached 1,156.4 mg L−1 was obtained with a high substrate concentration (40 mM 13-HPOT) under the following conditions: 10 min, pH 7.5, 20 °C, HPL 16 U mL−1, butylated hydroxytoluene (BHT) 1 mM, and dithiothreitol (DTT) 15 mM. Then the separation of (2E)-hexenal was conducted by salt-adding steam distillation. It was found that AlCl3 had the greatest effect on the separation of (2E)-hexenal, followed by CaCl2 and NaCl. The distillate yield with the addition of AlCl3 was 93.2 %, while the distillate yield without the addition of salt was 81.2 %. The distillate concentration with AlCl3 was 6,464.2 mg L−1, while the distillate concentration without the addition of salt was 4,490.3 mg L−1. The addition of salt improved the efficiency of the steam distillation. This study was greatly meaningful for providing a green method to the large-scale production of (2E)-hexenal.