K-ras oncogene is frequently found in human cancers and thus may serve as a potential diagnostic marker for cancer cells in circulation. So far, there is no reliable method for detecting cancer cells with K-ras oncogene in peripheral blood. The objective of this study was to develop a diagnostic membrane array using activated K-ras oncogene-associated molecules as detection targets. In our previous study, cDNA microarray analysis showed that there were 94 genes differentially expressed in K-ras mutant stably transfected adrenocortical cells. In the present study, we obtained 22 up-regulated genes in the closest relation to K-ras oncogene through bioinformatic analysis. At first, we carried out membrane array analysis by using in vitro culture cells. We demonstrated that this diagnostic technique was feasible and highly sensitive. A number as low as 5 cancer cells bearing K-ras oncogene in 1 ml of blood could be distinctively detected. Then, we collected blood specimens from 76 cancer patients. Direct sequencing analysis of these 76 samples showed that K-ras mutation was present in 43 patients with mutation sites mainly at codons 13, 15 and 61, which have been commonly established to be activated sites. We subsequently analyzed these 76 specimens with our diagnostic membrane array. Thirty-nine specimens were detected as positive for activated K-ras oncogene. Eighty percent (12/15) of mutations occurred at codon 13, 72.7% (8/11) at codon 61, and 88.9% (8/9) at codon 15 were accurately detected by our diagnostic membrane. Finally, through a series of biostatistical analyses, the sensitivity, specificity and accuracy of the diagnostic membrane array were 83.7, 90.9 and 86.8%, respectively. These findings suggest that the K-ras oncogene membrane array has a great potential for further investigation and clinical application.