Twenty-four new 1,3-disubstituted urea derivatives were synthesized and reported for the first time. The antiproliferative activities of these compounds were evaluated against a panel of one human liver cell line (L02) and two human tumor cell lines (KB and K562) by applying the MTT colorimetric assay. The series of 1,3-disubstituted urea derivatives show good antiproliferative activity against human cancer cell lines (KB and K562) and no antiproliferative activity against liver cell line (L02). The potent in vitro antiproliferative activity of these derivatives and their selectivity for L02 are quite important points for an anticancer drug candidate with fewer side effects. Structure-activity relationships were also discussed based on the obtained experimental data. The hydroxyl groups on the phenyl ring reduced the antiproliferative activities of 1,3-disubstituted urea derivatives. The OH groups could be responsible for a reduction in the permeability of the cell membrane. Generally, an aromatic ring on N-3 seems to be in favor of enhancing the inhibitory activity, compounds introduced a nitro group substituent at C-3 position on the aromatic ring approved to generally decrease activity.