The aim of this study was to present a non-trypsin 3D cell culture method with a reversible thermosensitive HBCS hydrogel. In this study, hydroxybutyl chitosan (HBCS) was synthesized by grafting hydroxybutyl groups on chitosan molecule chains. The prepared HBCS was water-soluble, and the reversible phase transformation temperature was 26 °C. Scanning electron microscope images illuminated the 3-D network of hydrogel formed irregular porous structure which ranged from 50-250 μm. Cell viability assay indicated that HBCS solution could promote the proliferation of human umbilical vein endothelial cells (HUVECs), and the boost of proliferation was enhanced with the increase of HBCS concentration. HBCS had no harm to the nitric oxide (NO) synthesis functionality of HUVECs. HUVECs could grow and reproduce inside the hydrogel, and showed good vitality after 14-days culture. Meanwhile, cells cultured inside the hydrogel could be passaged successively through the reversible phase transformation process of HBCS. The results revealed that HBCS have the potential to be used for 3-D cell culture without the use of trypsin.