In this work, a three-dimensional tumor-microvascular structure was simulated on a microfluidic chip for study of antioxidants effects on malignant glioma cells in vitro. The 3D hydrogel containing lumen was constructed to co-culture endothelial cells and glioma cells to mimic tumor microvascular environment. Macroporous gelatin transglutaminase (TG) hydrogel was prepared with biological and mechanical properties suitable for cells culture and nutrient refresh. To reform a vessel structure, U87 cells were dispersed in the TG-gelatin hydrogel and HUVEC cells were seeded in the lumen of hydrogel. Three typical antioxidants (α-lipoic acid, catechins and ascorbic acid) have been selected to research the antioxidant effects of glioma cells in the simulative tumor microenvironment. The results showed that the HUVEC cells formed vessel presented the transportation and penetrable functions for antioxidants from lumen to glioma cells. The antioxidants displayed higher selectivity to U87 cells than HUVEC cells and α-lipoic acid has a strong antioxidant capacity.