Objective To observe the effect of intraoperative and preoperative chemotherapy on the expression of p53, Ki-67 and prognosis in patients with advanced gastric cancer. Methods 40 cases of advanced gastric cancer in our hospital from Aug 2014 to May 2015 were enrolled in the study, and were divide into 2 groups randomly. In group I, 20 patients received chemotherapy intervention befoerer operation, and the other group received chemotherapy intervention during operation. The expressions and prognosis of p53 and Ki-67 were observed and compared between the two groups. Results Group Ⅰ and group Ⅱ after treatment, p53 and Ki-67 were higher than that before treatment, with statistical significance(P<0.05). However, there was no significant difference in the expression of p53 between group Ⅰ and group Ⅱ after treatment, and there was no significant difference(P>0.05). Before and after treatment, the difference of AI was significant, with statistical significance (P<0.05). The effect of group Ⅰ was obviously better than that of group Ⅱ, the difference was statistically significant(P<0.05). Six monthse after the operation and one year follow-up found two groups of recurrence rate and mortality rate had no significant difference(P>0.05). After two years follow-up found the group Ⅱ recurrence rate, mortality was lower than in group Ⅰ (P<0.05). Conclusion The effect of intraoperative chemotherapy is better than that of preoperative chemotherapy, and the prognosis is better than that of preoperative chemotherapy.