Objective To explore the effect of decoction of Euphorbia fischeriana Steud. and jujuba (DEFSJ) against estrogen receptor (ER) negative (-) and ER positive (+) of breast cancer via PI3k/Akt pathway, and to provide reference for targeted treatment of breast cancer. Methods DEFSJ extract was prepared and analysed by using UHPLC-Triple Quad. DEFSJ containing serum (CS) was prepared by a method of serumal pharmacology. Different concentrations of DEFSJ-CS were applied to (ER-) MDA-MB-453 cells and (ER ) MCF-7 of breast cancer in vitro for 48 h. Flow cytometry was used to detect the distribution of cellular cycle，DNA ladder assay was used to assess the degree of apoptosis，and Western blot was used to detect the expression of PI3k/Akt pathway-related proteins. The expressions of FoxO3a, FoxO1a and Bim mRNA were detected by a real-time quantitative polymerase chain reaction (qrt-PCR) method. Nuclear translocation of FoxO3a protein was detected by a confocal laser microscopy. Results Five batches of the DEFSJ extract were analysed by using UPLC, and the results showed that the preparation technology was feasible and the quality was controllable, ensuring the accuracy of pharmacological experiments results. DEFSJ-CS can block cells in G2/M phase (P<0.05，P<0.01). The cells treated with DEFSJ-CS emerged the typical apoptotic ladder in DNA ladder experiment. Compared with negative control group, DEFSJ-CS can decrease the proteins expression of p-PI3k，p-Akt，p-FoxO3a and p-FoxO1a (P<0.05, P<0.01) and can increase the proteins expression of Bim (P<0.05, P<0.01), and can decrease the mRNAs expression of FoxO3a and FoxO1a (P<0.05, P<0.01) and can increase the mRNA expression of Bim (P<0.05, P<0.01), and can enhance nuclear translocation of FoxO3a protein in the cells. Besides, these datas all showed that DEFSJ-CS had better effect on (ER-) MDA-MB-453 cells than (ER ) MCF-7 cells. Conclusion The regulatory effect of the DEFSJ extract on anti-breast cancer is involves the PI3k/Akt pathway, and the effect is varies with phenotypic differences.