The invention provides a detection kit and a detection method for human BRAF gene V600E mutation. The detection kit comprises a first reagent pack and a second reagent pack, wherein each of the first reagent pack and the second reagent pack comprises PCR buffer solution, dNTP, MgCl2, a specific probe, a locked nucleic acid (LNA) primer, an interior label system, HotStart Taq enzyme and UNG enzyme; the first reagent pack comprises a primer pair SEQ ID NO:1 and SEQ ID NO:2, and a probe SEQ ID NO:3; the second reagent pack comprises a primer pair SEQ ID NO:1 and SEQ ID NO:4, and a probe SEQ ID NO:5; and the SEQ ID NO:4 is LNA primer. The detection kit and the detection method can be used for detecting human BRAF gene V600E mutation, and has the advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high flux, safety, objective result determination and the like.