Background: Immunotherapy has been explored as a new therapy for B cell lymphoma, which is a non-Hodgkin's lymphoma. Because CD20 is a B lymphocyte-specific marker, anti-CD20 single chain-tagged T lymphocytes have already begun to be experimentally used in B cell lymphoma treatment, but its use is still limited because of its unspecific targeting. T cells transfected with CD28 and CD137 can significantly improve the ability of cytokines secretion and anti-tumor effect, as well as extending T cell survival time and improving their proliferation ability. Material/Methods: Genes containing anti-CD20-CD28-CD137-TCR zeta were constructed. After cloning and sequencing, the plasmid was constructed and packaged by lentivirus. It was transfected to the peripheral blood T lymphocyte after identification transfection to induce the fusion protein expression. The cells were incubated with Raji cells and the LDH test was performed to detect the cytotoxic effect of CAR-T cells; the tumor volume and survival rate were measured to observe its inhibitory effect on B cell lymphoma in nude mice. Results: Gene with anti-CD20-CD28-CD137-TCR zeta was successfully constructed and transfected to the T cell surface. LDH assay revealed that CAR-T cells can kill the Raji cells with a killing rate of 32.89 +/- 6.26%. It can significantly inhibit B cell lymphoma growth in nude mice. Conclusions: T lymphocytes transfected with anti-CD20-CD28-CD137-TCR zeta fusion gene can kill B cell lymphoma, which could provide a new strategy for tumor treatment.