The prevalence of cancer in companion dogs is growing nowadays with the increasing worldwide population of domestic dogs. Since there is a less established standard of care in veterinary medicine, investigational treatments, such as the development of biomarkers can be considered as a therapeutic intervention for early diagnosis. Despite the enormous efforts that have been invested in the search of biomarkers, still, there is a need for easy detection of significant biological markers for predicting canine cancers at an early stage. In this study, we have analyzed the expression pattern of previously reported 46 canine cancer-associated candidate genes in blood specimens using real-time qPCR. We hypothesized that analysis of gene expression in blood would provide preliminary evidence of local or systemic immunogenic response which further contribute to the easy and early diagnosis of canine cancer from blood specimen as an analytical tool. The datasets included a total of 22 blood samples collected from different breeds of dogs diagnosed with cancer and five from healthy normal dogs. RT-qPCR analysis was performed by employing the SYBR Green PCR mix to assess the expression of these 46 genes in a total of 27 samples. From our result, a total of nine genes (ROS1, C1QA, CD48, IL1b, TLR2, IL2R, CHI3L1, CTSS, and TLR7) were found to be significantly up-regulated (p < 0.05 and p < 0.01) in the cancer samples compared to non-cancer samples. The relative expression level of ROS1, C1QA, CD48, IL1b, TLR2, IL2R, CHI3L1, CTSS, and TLR7 genes was 5.74, 4.78, 3.94, 2.94, 2.57, 2.53, 2.50, 2.04, and 2.57, respectively, in cancer samples compared to non-cancer samples. Thus, our results reveal several highly expressed cancer genes that can be therapeutic target genes for further testing in canine cancers.