Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence 'Eprobe' capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05-0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.