K-ras gene mutation is generally regarded as a significant indicator for colorectal cancer screening and early diagnosis and in favor of filtering out colorectal cancer patients benefiting from epidermal growth factor receptor (EGFR)-targeted therapy. The chip-based temperature gradient capillary electrophoresis (TGCE) was successfully used to detect K-ras gene mutation in 98 cases of paraffin-embedded colorectal cancer tissues with high sensitivity. Mutation-positive detection rate from chip-based TGCE (47.96%) was significantly higher than that from the PCR product direct sequencing (23.47%). Clone sequencing showed that this detection system could detect at least 2.08% of the mutant K-ras gene. Analysis of the relationship between K-ras gene mutation and clinical pathological parameters revealed that K-ras gene mutation rate in rectum cancer was significantly higher than that of colon cancer (P < 0.05), but no remarkable correlation between K-ras gene mutation rate with age, gender, histological type and tumor stage was observed. Therefore chip-based TGCE is very sensitive and rapid method for mutation detection, and it can be applied for large-scale screening, early diagnosis and guiding clinical treatment.