As the continuation of our work on the development of tubulin inhibitors with potential anticancer activities, novel bis-substituted aromatic amide dithiocarbamate derivatives were designed by contacting bis-substituted aryl scaffolds (potential anti-tubulin fragments) with N-containing heterocycles (potential anti-tubulin fragments) in one hybrid using the anticancer dithioformate unit as the linker. The antiproliferative activity against three digestive tract tumor cells was evaluated and preliminary structure activity relationships were summarized. Among these compounds, compound 20q exhibited most potent antiproliferative activity against MGC-803, HCT-116, Kyse30 and Kyse450 cells with IC values of 0.084, 0.227, 0.069 and 0.078 μM, respectively. In further studies, compound 20q was identified as a novel tubulin inhibitor targeting the colchicine binding site. Compound 20q could inhibit the microtubule assembly and disrupt cytoskeleton in Kyse30 and Kyse450 cells. The results of molecular docking suggested that compound 20q could tightly bind into the colchicine binding site of tubulin by hydrogen bonds and hydrophobic interactions. Compound 20q dose-dependently inhibited the cell growth and colony formation, effectively arrested cells at the G2/M phase and induce mitochondrial apoptosis in Kyse30 and Kyse450 cells. In addition, Compound 20q could regulate the expression of G2/M phase and mitochondrial apoptosis related proteins. Collectively, compound 20q was here reported as a novel tubulin inhibitor with potential anticancer activities.