Preparation of oligonucleotide microarray for gene expression detection of tumor-associated gene and its primary application in study on molecular mechanism of antisense oligonucleotide “cantide” were carried out. An oligonucleotide microarray consisting of about 450 kinds of tumor-associated genes was constructed and the standards of quality controls of it were founded at the same time. Tumor cells were treated with “cantide” mixed with lipofectin and total RNA was extracted. Through reverse-transcription, cDNA was synthesized and labeled with Cy5 or Cy3. After hybridized with the prepared microarray, signal intensities were detected and expression profiles were analyzed by software. The results indicate that the oligonucleotide microarray prepared has high specificity,good sensitivity and stablity etc. Compared to cells treated with lipofectin alone, the mRNA expression levels of seven genes appeared to show significant change. The mRNA expression of MDNCF , DHS genes were downregulated,while the mRNA expression of MUC2,MPP11,LAT,HRIF-B,JNK3A1 genes were upregulated after HepG2 cells treated with “cantide” for 15 h. The data should provide some candidate genes that may be involved in antitumor effect of “cantide”, and it is helpful to make a further investigation on the molecule mechanism. It can be concluded that the prepared oligonucleotide microarray can be used to investigate the gene expression profile of tumor-associated genes and provide techonical paltform for clinical diagnosis and basic research.