The invention relates to the field of refined extraction, in particular to a preparation method of aristololactam new compound and application thereof. The preparation method is prepared from the following steps of performing alcohol extraction on Aristolochia contorta Bunge. and preparing extractum; utilizing 2.5L of distilled water to fully dissolve and dilute the obtained Aristolochia contorta Bunge. alcohol extractum, extracting through 2L of chloroform, extracting 3 times and reducing pressure and concentrating extract liquor to obtain corresponding chloroform extractum; eluting the chloroform extractum through a silica gel column chromatography (petroleum ether to ethyl acetate is equal to (100 to 0):(0 to 100), combining through thin layer chromatography silica gel plate spotting and dividing the same cut fraction as one group to obtain 13 groups of Fr1-13; eluting cut fraction Fr5 through silica gel column, obtaining Fr5-2 through petroleum ether to acetone as 92 to 8, obtaining cut fraction Fr5-2-3 through LH-20 gel column elution, then eluting through a silica gel column, utilizing petroleum ether-ethyl acetate as an eluting system and obtaining purified compound M4 when a volume ratio is 65 to 35. The Aristolochia contorta Bunge. is fully utilized, and M1, M4 and M6 have very good sterilizing activity.