The invention relates to a method for separating and determining <238-240>Pu, <241>Am and <90>Sr in a low-radioactivity sample, which comprises the following steps: (1) sample pretreatment; (2) separation and enrichment of <238-240>Pu, <241>Am and <90>Sr: after ascorbic acid and sodium nitrite are added into a solution obtained in step (1) to regulate the valence state of plutonium into tetravalence, the solution passes through a TEVA, TRU and SR tandem extraction chromatography resin column, and the flow velocity is kept at 1mL / min to 1.2mL / min; at the moment, <238-240>Pu is adsorbed on the TEVA column, <241>Am is adsorbed on the TRU column, and <90>Sr is adsorbed on the SR column; 20mL to 25mL of hydrochloric acid solution which is 0.02mol / L is used for eluting <238-240>Pu on the TEVA column, 15mL to 20mL of hydrochloric acid solution which is 3.0mol / L is used for eluting <241>Am on the TRU column, and 15mL to 20mL of nitric acid solution which is 0.01mol / L is used for eluting <90>Sron the SR column; (3) determination of <238-240>Pu, <241>Am and <90>Sr: a cerium fluoride micro-deposition method is adopted to prepare an Alpha source, and the <238-240>Pu eluent and the <241>Am eluent are measured on the Alpha energy spectrum; the <90>Sr eluent is placed for 14 days, and after <90>Sr<-><90>Y is balanced, determination is carried out by the Cerenkov counting method. The analysismethod can be adopted to separate and enrich Pu, <241>Am and <90>Sr in a sample, and the period of analyzing Pu, <241>Am and <90>Sr is shortened.