Disclosed are a folded chlorotoxin, a chlorotoxin variant and a folded chlorotoxin variant and their preparation technology. The folded chlorotoxin has a peptide sequence of MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR-NH2, and the folded chlorotoxin variant has a peptide sequence of MCMPCFTTDHQMARSCDDCCGGSGRGSCYGPQCLCR-NH2 and is formed by replacing serine (Ser, S) by lysine (Lys, K) in the peptide sequence of chlorotoxin. The chlorotoxin and its derivatives have potential application values in biological and medical fields and good economic and social benefits to life, health, and personalized healthcare.