The invention discloses a demulsifying agent and a preparation method thereof. The invention further discloses a demulsification method of hydrocarbon oil by adopting the demulsifying agent. The demulsifying agent contains at least one compound represented by a formula I. The demulsifying agent disclosed by the invention has a good demulsification effect on hydrocarbon oil, especially acid-containing crude oil; high dewatering efficiency can be obtained; and the oil content in separated water can be effectively reduced.