Lymphotoxin(LT) delivers the signal of apoptosis by TNFR1, sequentially develops anti-tumor activity. Several receptor binding sites of LT were mutated randomly, then the R46 ， S106 ， L130 combined site-directed random mutation library and S106 ～ F110 region random mutation library were displayed on a filamentous phage surface. By using TNFR1 as solid phase molecule, the phage libraries were biopanned and positive mutant clones were enriched. 20 monoclonal phages were picked randomly to detect receptor binding by ELISA assay, 80% of them can bound TNFR1 specifically, and TNFR1 binding ability of 4 clones was higher than rhLT with wild type sequence. 4 mutants with high binding ability were subcloned to pET32a(+) vector, after expression in E.coli and purification process, the binding ability and the cytotoxicity in L929 mouse fibroblast cells of these mutants were examined. 3 mutants were having similar binding ability compared with monoclonal phage, the binding ability to TNFR1 and the cytotoxicity to L929 of C199 clone have been enhanced near 30% and 90% respectively. The effort to proceed molecular evolution study of lymphotoxin in vitro by phage display not only can be very useful for the following research involved in the relation between structure and function, but also provides potent tool for macromolecule drug development