This study was aimed to provide references for techniques in future application of traditional and natural drugs for tumor prevention and treatment. Domestic and foreign literatures on applications of gene chip technology in antitumor studies with natural and traditional medicine from 2000 to 2014 were reviewed. Corresponding database was established from aspects of published articles, drug intervention types, study fields, sample sources, chip technology platforms, repeatability of gene chip experiments, criteria of differential genes, and validation of gene chip experiments. Application experiences of gene chip in antitumor natural and traditional drugs were summarized. Shortcomings of gene chip technology application were analyzed deeply. The results showed that experimental gene screening was limited at the cellular level. More attentions should be paid to experiments at the animal and clinical levels. Scholars had paid more attention to expression level of mRNA and less attention to gene regulation level and epigenetic research of microRNA chip and DNA methylation chip. Gene chip experiments were lack of repeatability, which directly affected the evaluation results of difference gene and reduced reliability of gene screening. Screening results of genes should be verified not only at the mRNA level, but also increased at the protein level. It was concluded that gene chip was one of the most mature technologies to detect the level of gene expression, which was widely used in the research field of traditional and natural antitumor drug studies. Researchers should try to avoid deficiencies mentioned above in experiments related to gene chip technology.