A series of novel bivalent β-carbolines were synthesized and evaluated for anti-proliferative activities on a panel of cancer cell lines, apoptosis induction and cell cycle effects. The present study showed that bivalent β-carbolines possess stronger anti-proliferative effects than monomer. Moreover, introduction of a methyl or benzyl group into position-N9 of bivalent β-carbolines improved their anti-proliferative activities. Additionally, the most potent compounds 4c and 8a with IC50 values of 0.84 µM and 0.23 µM respectively, were more potent than doxorubicin (IC50 = 2.48 µM) against A-549 cell lines. The most active compound 8a could induce cell apoptosis in a dose-dependent manner and cause cell cycle arrest in the S phase.