Disclosed is a Madin-Darby canine kidney (MDCK)-derived cell line. The MDCK-derived cell line is derived from MDCK cells deposited under accession number ATCC CCL-34. The MDCK-derived cell line can be prepared by serum-free culture and suspension culture. Preferably, the MDCK-derived cell line has low or no tumorigenicity. The MDCK-derived cell line is preferably selected from MDCK Sky1023, MDCK Sky10234 and MDCK Sky3851. Further disclosed are a culture method for growing the MDCK-derived cells and a method for producing a vaccine virus using the MDCK-derived cells.