Objective 1-methoxycarbonyl-β-carboline, an active ingredient exists in a number of Chinese herbs, has anti-tumor, anti-inflammation and other biological activities, but its target and mechanism are unclear. In this paper, the action target of 1-methoxycarbonyl-β-carboline was studied in order to elucidate its action mechanism. Methods MTT assay was used to detect the effects of 1-methoxycarbonyl-β-carboline on the viability of various tumor cells. ADP-Glo kinase assay was used to detect the effects of 1-methoxycarbonyl-β-carboline on 217 kinases. The interaction between 1-methoxycarbonyl-β-carboline and protein kinase MLK3 was elucidated by molecular docking experiment. Results 1-methoxycarbonyl-β-carboline had certain anti-tumor proliferation activity against various tumor cells. At the concentration of 10 μM, the inhibition rate of 1-methoxycarbonyl-β-carboline on 22 kinases was more than 50%. Among them, MLK3 showed the highest inhibition rate of (88.41%). Molecular docking experiments showed that 1-methoxycarbonyl-β-carboline could occupy the ATP-active cavity of MLK3 and form hydrogen bond with the key amino acid residue ALA-196 in its hinge region. Conclusion 1-methoxycarbonyl-β-carboline may exert anti-tumor pharmacological activity by inhibiting MLK3 and other kinase targets