A thermo-sensitive hydrogel composed of chitosan and α, β-glycerophosphate was successfully synthesized and used as basic substrate for primary culture and subculture of lymphoid cells from shrimp, Penaeus chinensis. When the ambient temperature increases from 4°C to 25°C, the chitosan/α, β-glycerophosphate solution transforms into a semi-solid three-dimensional hydrogel with a rough surface. Primary lymphoid cells formed were round and grew homogenously over the surface of the hydrogel. The peroxidase activity indicated that cells seeded on the hydrogel suffered less stress than cells suspended in control culture flasks. The higher relative viability and alkaline phosphatase activity of primary cultured lymphoid cells indicated that the chitosan/α, β-glycerophosphate hydrogel enhanced the growth and proliferation of lymphoid cells. Subsequent subculture of lymphoid cells starting from crumbled primary hydrogel culture was also successful.