4-anilinoquinazoline-based derivatives represent an attractive scaffold for small molecular EGFR-TKIs in the field of medicinal chemistry. A series of novel heterocyclic substituted derivatives have been designed, synthesized and evaluated their antitumor bioactivities as potential EGFR-TKIs. Most of the new compounds exhibited certain efficient inhibition potency for proliferation of a panel of five human cancer cells with IC50 values at the low micromolar level, and some of them possessed good broad-spectrum inhibition activities, compared to Gefitinib. Especially, the IC50 values of compound 21 against HepG2, A549, MCF-7, DU145 and SH-SY5Y cells were 4.61, 9.50, 9.80, 6.79 and 7.77 μM, respectively, which were much lower than those of Gefitinb. Furthermore, the highlighting compound 21 demonstrated excellent inhibition activity against EGFR-TK with the IC50 value of 3.62 nM, similar to that of Gefitinib(2.21 nM). The results of LDH release assay proved that compound 21 was anti-proliferative rather than cytotoxicity on HepG2 cells. Compound 21 were able to cause HepG2 cells to block in S phase and induce cell death mainly by apoptosis through a mitochondrial dependent pathway. Moreover, the assessment of MMP, the determination of intracellular free Ca2+ concentration, the production of ROS, and the effects on the activity of caspase-3 in a dose-dependent manner demonstrated that compound 21 induced cell apoptosis in HepG2 cells through the Ca2+/ROS-mediated mitochondria/caspase-dependent apoptosis pathway largely. These preliminary results evidenced that compound 21 could be a potential antitumor agent deserving further study.