Inhibition of tubulin polymerization is one of the significant strategies in the treatment of cancer. Inspired by the excellent antitumor activity of EP128495 and the beneficial biological activities of selenium compounds, a series of new selenium-containing 4-anilinoquinazoline hybrids were synthesized and evaluated as tubulin polymerization inhibitors. An anti-proliferative activity assay showed that most of the compounds inhibited human sensitive cancer cells at low nanomolar concentrations. A mechanism study revealed that the optimal compound 5a disrupted microtubule dynamics, decreased the mitochondrial membrane potential and arrested HeLa cells in the G2/M phase, finally resulting in cellular apoptosis.